DEAH-box proteins

ABSTRACT

The present invention relates to the identification and molecular-biological and biochemical characterization of novel DEAH-box proteins which show structural similarity with other known helicases. The invention also relates to DNA sequences encoding such proteins. It is demonstrated that such proteins bind ATP and nucleic acid and possess helicase and ATPase activities. This invention further relates to processes for preparation of such proteins and their use in pharmacologically relevant test systems and therapeutic applications.

BACKGROUND OF THE INVENTION

The modulation of RNA structure is an essential regulatory process inmany cellular events, such as, for example, pre-mRNA splicing, assemblyof spliceosomes, assembly of ribosomes, protein translation, which canbe summarized under the generic term "regulation of gene expression atthe RNA level". The so-called "DEAD box" protein family of putative RNAhelicases, named after the characteristic amino acid motifAsp-Glu-Ala-Asp (in the single-letter code DEAD) (SEQ ID NO:35), in thiscontext plays a key part (in particular for the modulation of thesecondary and tertiary structure of mRNA). DEAD box proteins are alsoinvolved in processing of DNA. The members of this family and somesubfamilies have differences in their specific function and cellularlocalization. However, in addition to characteristic sequence homologiescertain members also show similar biochemical properties (F. V.Fuller-Pace, Trends in Cell Biology, Vol 4, 1994, 271-274). Thecharacteristic protein sequences of the DEAD proteins are highlyconserved in evolution (S. R. Schmid and P. Lindner, Molecular andCellular Biology, Vol 11, 1991, 3463-3471). Members of this proteinfamily are found in various viruses, bacteria, yeasts, insects, molluscsand lower vertebrates up to mammals and are responsible for a largenumber of cellular functions. The fact that even relatively simpleorganisms such as, for example, the yeast Saccharomyces cerevisiaeexpress numerous proteins of the DEAD box protein family and theirsubfamilies, suggests that each of these proteins contributes to thespecific interaction with certain RNAs or RNA families (I. lost and M.Dreyfus, Nature Vol 372, 1994, 193-196). It has been shown thattranslation factors, such as elF-4A and the proteins involved in thepre-mRNA splicing process, recognize specific RNA target sequences orstructures. Nevertheless, to date there is little information about thestructure and the synthesis of characteristic RNA sequences whichrequire the DEAD proteins for recognition and for ATPase/RNA helicasereaction (A. Pause and N. Sonenberg, Current Opinion in StructuralBiology Vol 3, 1993, 953-959).

The DEAD box protein family is an enzyme class which is growing andwhich is involved in the various reactions in post transcriptionalregulation of gene expression. Because of the high number of differentcellular DEAD box proteins, it is to be expected that specific RNAhelicases are assigned to certain classes of gene products, e.g. viralproteins, heat shock proteins, antibody and MHC proteins, receptors,RNAs etc. This specificity indicates that members of this protein familyare attractive pharmacological targets for active compound development.

Two of the subclasses of the DEAD (SEQ ID NO:35) box protein family arethe DEAH (SEQ ID NO:36) proteins (having one specific amino acidreplacement) and the DEXH (SEQ ID NO:37) protein (having two amino acidreplacements in the main motif, X being any desired amino acid)families, which also play a part in the replication, recombination,repair and expression of DNA and RNA genomes (Gorbalenya, A. E., Koonin,E. V., Dochenko, A. P., Blinov, V. M., 1989: Nucleic Acids Res. 17,4713-4729). The DEAD box proteins and their subfamilies are oftendesignated "helicase superfamily II " (Koonin, E. V., Gorbalenya, A. E.,1992: FEBS 298, 6-8). This superfamily has seven highly conservedregions. Altogether, up to now over 70 members belong to thissuperfamily II.

The following schematic representation of the DEAD family and the DEAHand DEXH families subfamilies (Schmid, S. R., Lindner P., 1991:Molecular and Cellular Biology 11, 3463-3471) shows the similaritybetween the families. The structure of elF-4A, a member of a DEAD boxprotein, is also shown. The numbers between these regions show thedistances in amino acids (AA). X is any desired AA. Where known,functions have been assigned to the ranges.

                       DEAD (SEQ ID NO:35) FAMILY                                                             ATPase A motif                    ATPase B                           motif                                                                         NH.sub.2 -----AXXXGKT----PTRELA-----GG---TPGR---DEAD---                       SAT---FXXXT---                                                                   21-299      24-42       22-28  19-27   19-22                               27-51  59-70     52-53                                                        RGXD-----HRIGRXXR---COOH (SEQ ID NO:19)                                              20           24-236                                                    elF-4A                                                                        NH.sub.2 -----AXXXXGKT---PTRELA-----GG---TPGR---DEAD---                       SAT---FINT---                                                                      75           24           22    20      20                               27      62   52                                                               RGID------HRIGRXXR---COOH (SEQ ID NO:20)                                             20                41                                                   DEAH (SEQ ID NO:36) SUBFAMILY                                                 NH.sub.2 -----GXXXXGKT---RVAA-----XX---TDGX---DEAH---SA                       T---FXT---                                                                       245-505         22-24     29   7-8     19       28                            58-61   75-84                                                              XGXX------QRIGRXGR---COOH (SEQ ID NO:21)                                             25           315-373                                                   DEXH (SEQ-ID NO:37) SUBFAMILY                                                 NH.sub.2 -----XXXXXGKT---PTRXXX----------------DEXH---T                       AT---FXXS---                                                                     81-1904            19-27      55-60                                        24-30  44-72                                                                    46-55                                                                       XGXX------QRXGRXGR---COOH (SEQ ID NO:22)                                            38-44        155-1799                            

The ATPase motif (AXXXXGKT, SEQ ID NO:23) is an amino-terminal conservedregion and occurs in most proteins which bind nucleotides, i.e. also inother proteins which interact with DNA and RNA, such as DNAB (part ofthe primosome), UvrD (endonuclease), elongation factor 1 andtranscription termination factor Rho (Ford M. J., Anton, I. A., Lane, D.P., 1988: Nature 332, 736-738). As used in this specification "ATPaseactivity" is used to mean the ability to catalyze hydrolysis of ATP. TheATPase A and ATPase B motifs function together in the enzymatic processof ATP hydrolysis.

The second conserved region is the so-called DEAD (SEQ ID NO:35) box, orDEAH (SEQ ID NO:36), DEXH (SEQ ID NO:37) or DEXX (SEQ ID NO:38) box inother families of the helicases and nucleic acid-dependent ATPases. Thisregion represents the ATPase B motif. In the reaction mechanism, theN-terminal aspartic acid in the DEAD box binds Mg²⁺ via a water molecule(Pai, E. F., Krengel, U., Petsko, G. A., Gody, R. S., Katsch, W.,Wittinghofer, A., 1990: EMBO J. 9, 2351-2359). Mg²⁺ in turn forms acomplex with the β- and gamma-phosphate of the nucleotide and isessential for the ATPase activity. Substitutions of the first two aminoacids of the DEAD region in elF-4A prevent ATP hydrolysis and RNAhelicase activity, but not ATP binding (Pause, A., Sonenberg, N., 1992:EMBO J. 11, 2643-2654). The DEAD region additionally couples RNAhelicase activity to ATPase activity. The hydrolysis of ATP provides theenergy needed for RNA unwinding during helicase activity.

The third region investigated is the SAT region (sometimes also TAT). Asa result of mutation in this region, RNA helicase activity issuppressed, but other biochemical properties are retained (Pause A. &Sonenberg N., 1992). As used in this specification "helicase activity"is used to mean the ability to directly or indirectly catalyze theunwinding of RNA.

The farthest carboxy-terminal region is the HRIGRXXR (SEQ ID NO:24)region, which is necessary for RNA binding and ATP hydrolysis.

As stated above, members of the DEAD box protein family bind ATP andnucleic acid. As used in this specification a protein that "bindsnucleic acid" is defined as a protein that forms complexes with nucleicacid. The binding can be measured by standard methods likeElectrophoretic Mobility Shift Assay (EMSA) or ELISA, which are wellknown in the art. The following assays may also be used: ScintillationProximity Assay (SPA, Amersham International, Little Chalfont,Buckinghamshire, England) and BlAcore (Biomolecule Interaction Analysis,Pharmacia, Upsala Sweden).

As used in this specification, a protein that "binds ATP" is defined asa protein that will bind ATP as measured using an assay that measuresability of labeled ATP to bind to protein. The ATP may be labeled usingradioactive or fluorescent label. One example of an ATP binding assay isdescribed in Pause, et al. EMBO J. 11:2643 (1992), which is herebyincorporated by reference. Briefly, a protein according to the inventionis incubated in a crosslinking reaction mixture containing Tris-HCl (pH7.5), Mg acetate, ³² P-ATP, glycerol and DTT in the presence or absenceof poly(u) (Pharmacia) under a 15 watt germicidal lamp at 4° C.Unlabelled ATP is then added, followed by addition of RNase A at 37° C.Samples are boiled in SDS-PAGE sample buffer and electrophoresed.

It follows from the above-mentioned relationships that specific RNAhelicases are attractive targets for pharmaceutically active substances.For example, it is also known that certain pathogenic viruses, which cancause diseases in humans, animals or plants, carry in their genome agene encoding an RNA helicase, which is needed for accurate replication(E. V. Koonin, 1991). Thus, specific substances that interfere with, ormodulate, the activity of such virus-specific helicases could be used totreat virally-mediated diseases. Because helicases are also found inplants, substances that modulate plant helicases may be used to protectplants against pathogenic viruses. (F. V. Fuller-Pace, Trends in CellBiology, Vol. 4, 1994, 271-274). Helicases also make attractive targetsfor development of therapeutic treatments for various types of diseases.For example, hereditary diseases such as Werner's syndrome and Bloom'ssyndrome have been linked to the production of proteins with helicasestructure. See Yu, et al. Science 272: 258 (1996) and Research News,Science 272: 193 (1 996)(Werner's); Ellis, et al. Cell 83:655 (1995),and D. Bassett "Genes of Medical Interest" Inhttp://www.ncbi.nih.gov/xREFdb/(Bloom's). A nucleolar RNA helicase isrecognized by the autoimmune antibodies from a patient with watermelonstomach. Valdez, et al., Nucl. Acid. Res., 24:1220 (1996). Inretinoblastoma cancer cells, expression of a DEAD box protein isamplified. Godbout, et al. Proc. Natl. Acad. Sci. USA 90:7578 (1993). Inaddition, RNA processing plays a role in a number of processes that areimplicated in other disease states. For example, in diabetic mice, theleptin receptor is abnormally spliced. Lee, et al. Nature 379:632(1996). In addition, post-transcriptional regulation of humaninterleukin-2 gene expression occurs at the level of processing ofprecursor transcripts, which may be linked to the presence of a protein.Gerez, et al. J. Biol Chem. 270:19569 (1995).

Thus, therapeutic agents can be designed that interfere with helicaseactivity or RNA processing that is associated with the disease state.

The isoxazole derivative leflunomide shows anti-inflammatory andimmunosuppressive properties without causing damage to the existingfunctions of the immune system (HWA486 (leflunomide); R. R. Bartlett, G.Campion, P. Musikic, T. Zielinski, H. U. Schorlemmer In: A. L. Lewis andD. E. Furst (editors), Nonsteroidal Anti-inflammatory Drugs, Mechanismsand Clinical Uses (Dekker: New York, 1994); C. C. A. Kuchle, G. H.Thoenes, K. H. Langer, H. U. Schorlemmer, R. R. Bartlett, R.Schleyerbach, Transplant Proc. 1991, 23:1083-6; T. Zielinski, H. J.Muller, R. R. Bartlett, Agents Action 1993, 38: C80-2). Many activities,such as the modification of cell activation, proliferation,differentiation and cell cooperation, which can be observed inautoimmune diseases, are modulated by leflunomide or its activemetabolite, A77 1726. ##STR1## Studies on the molecular mechanism ofaction of this active compound point to an influence on the pyrimidinemetabolism. Because leflunomide is very rapidly converted in the bodyinto A77 1726, in this specification, leflunomide and A77 1726 are usedinterchangeably. Thus, both "leflunomide resistance" and "A77 1726resistance" are used to designate the same condition.

Pyrimidine and purine nucleotides play a key part in biologicalprocesses. As structural units of DNA and RNA, they are thus carriers ofgenetic information. The biosynthesis of the pyrimidines comprises theoxidation of dihydroorotate to orotate, which is catalyzed by the enzymedihydroorotate dehydrogenase (DHODH). Altogether, six enzymes are neededfor the de novo synthesis of uridine monophosphate (UMP). UMP plays akey part in the synthesis of the other pyrimidines, cytidine andthymidine. The inhibition of DHODH thus leads to an inhibition ofpyrimidine de novo synthesis. Particularly affected are immune cells,which have a very high need for nucleotides, but can only cover a littleof this by side routes (salvage pathway). Binding studies withradiolabeled leflunomide analogs identified the enzyme DHODH as apossible site of action of A77 1726 and thus the inhibition of DHODH byleflunomide is an important starting point for the elucidation of theobserved immunomodulating activities. Williamson, et al. J. Biol. Chem.270:22467-22472 (1995).

SUMMARY OF THE INVENTION

In one embodiment, the invention provides an isolated DNA sequenceencoding a DEAH-box leflunomide-resistant protein. The invention alsoprovides such a DNA sequence wherein said protein has a molecular weightof 135 kilodaltons. The invention also provides such a DNA sequence,wherein said protein has a molecular weight of about 135 kilodaltons.

In another embodiment, the invention provides an isolated DNA sequenceas set forth in SEQUENCE ID NO. 15 (FIG. 8) and an isolated DNA sequenceas set forth in SEQUENCE ID NO. 17 (FIG. 9). In other embodiments, theinvention provides a homolog of the DNA sequence of SEQ. ID. NO. 15 anda homolog of the DNA sequence of SEQ. ID NO. 17.

In another embodiment, the invention provides isolated DNA sequencesencoding the amino acid sequence of SEQUENCE ID NO. 16 (FIG. 8) andencoding the amino acid sequence of SEQUENCE ID NO. 18 (FIG. 9).

In yet another embodiment, the invention provides an isolated DNAsequence that encodes a DEAH-box protein having one or more of thefollowing characteristics:

(a) the first homology domain (ATPase A, Domain I) is located more than650 amino acids from the N-terminus of said protein; (b) the N-terminusof said protein contains serine/arginine domains; (c) domain IV of saidprotein has the sequence FMP; (d) the distance between domains IV and Vof said protein is 74 amino acids or less; and (e) domain VI of saidprotein has the sequence QRSGRXGR (SEQ ID NO:25).

The invention also provides an expression vector comprising a DNAsequence according to the invention. The invention further provides ahost comprising such an expression vector. The invention also providesan antisense expression vector comprising a DNA according to theinvention, wherein said DNA sequence is inserted in reverse orientationinto said vector.

In another embodiment, the invention provides an isolatedleflunomide-resistant DEAH-box protein. The invention also provides sucha protein wherein said protein has a molecular weight of 135kilodaltons. The invention further provides such a protein wherein saidprotein has a molecular weight of about 135 kilodaltons. The inventionalso provides a mammalian protein, a protein isolated from a cell linederived from the murine cell line A20.2J and a human protein.

In yet other embodiments of the invention there is provided a proteincomprising the amino acid sequence of SEQUENCE ID NO. 16 (FIG. 8), or afragment thereof, or the amino acid sequence of SEQUENCE ID NO. 18 (FIG.9), or a fragment thereof.

In yet another embodiment, the invention provides an isolated DEAH-boxprotein having one or more of the following characteristics: (a) thefirst homology domain (ATPase A, Domain I) is located more than 650amino acids from the N-terminus of said protein; (b) the N-terminus ofsaid protein contains serine/arginine domains; (c) domain IV of saidprotein has the sequence FMP; (d) the distance between domains IV and Vof said protein is 74 amino acids or less; and (e) domain VI of saidprotein has the sequence QRSGRXGR (SEQ ID NO:25).

In another embodiment, the invention provides a process for thepreparation of a DEAH-box leflunomide-resistant protein, wherein saidprocess comprises:

(a) culturing a host cell comprising a vector encoding a DEAH-boxleflunomide-resistant protein and

(b) isolating said protein from the cell of step (a).

In yet another embodiment, the invention provides an "identifying"method for identifying a substance having one or more of the followingproperties: anticarcinogenic, anti-atherosclerotic, immunosuppressive,antiinflammatory, antiviral, antifungal or antibacterial, comprising:

(a) crystallizing a protein according to the invention;

(b) determining the three-dimensional structure said protein; and

(c) identifying said substance using molecular modeling techniques,wherein said substance affects the ability of said protein to bind ATPor nucleic acid.

The invention further provides such an identifying method wherein themethod comprises the additional step of determining the ability of theidentified substance to modulate the helicase activity of said DEAH-boxleflunomide-resistant protein. The invention also provides such anidentifying method comprising the additional step of determining theability of the identified substance to modulate the ATPase activity ofsaid protein. Finally the invention provides such an identifying methodcomprising the additional step of determining the ability of theidentified substance to modulate the splicing activity of said protein.In another embodiment, the invention provides a substance identifiedusing any of the foregoing methods.

In yet another embodiment, the invention provides a therapeutic methodfor the treatment of a disorder selected from the group consisting ofAlzheimer's disease, cancer, rheumatism, arthrosis, atherosclerosis,osteoporosis, acute and chronic infectious diseases, autoimmunedisorders, diabetes or organ transplant rejection, comprisingadministration of a pharmaceutically effective amount of a substanceidentified using the above-mentioned method to a patient in need of suchtreatment.

The invention further provides an "identifying" method for identifying asubstance that will modulate the helicase activity of a proteinaccording to the invention, comprising the additional steps of: (a)transforming a non-leflunomide-resistant cell with a DNA sequenceencoding a DEAH-box protein which binds nucleic acid and ATP, and whichhas helicase activity and ATPase activity, wherein the level ofexpression of said protein is significantly higher in aleflunomide-resistant cell than in a non-leflunomide-resistant cell,wherein said transformed cell is rendered resistant to leflunomide;

(b) culturing the cells in the presence of a high level of leflunomide;

(c) determining the ability of said substance to make the cells of step(b) non-leflunomide-resistant, wherein a substance that makes said cellsnon-leflunomide-resistant modulates the helicase activity of saidprotein.

In another embodiment, the invention provides a method for isolation ofRNA that binds specifically to a protein according to the invention,comprising:

(a) binding said protein or a fragment thereof to an affinity matrix;

(b) mixing an RNA sample to the matrix of step (a); and

(c) determining which RNA is specifically bound to said matrix. Theinvention also provides such a method comprising the additional step ofamplifying the RNA bound to said matrix by using the PCR technique. Theinvention also provides such a method, wherein said RNA of step (c) issubjected to sequence analysis.

Finally, in another embodiment, the invention provides a method forselecting a cell that contain heterologous DNA comprising:

(a) transforming cells with a vector comprising a DNA sequence encodinga DEAH-box leflunomide-resistant protein;

(b) growing said cells in the presence of a high level of leflunomide;and

(c) selecting a cell that will grow in the presence of said high levelof leflunomide; wherein said cell of step (c) contains said heterologousDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: SDS-PAGE (12% acrylamide). The left three gel traces are from aCoomassie Blue-stained gel, the three right gel traces from asilver-stained gel. M: Marker (Combithek from Boehringer Mannheim);A20.2J: normal A20 cells; A20R: A20 cells which are resistant to 100 μMleflunomide. In the Coomassie Blue-stained gel, 100 μg of protein ineach case were applied per gel pocket, in the silver-stained gel 5 μg ofprotein in each case. The arrow marks the protein which is expressed toan increased extent in resistant A20 cells.

FIG. 2: Peptide separation by HPLC. The HPLC was carried out accordingto the conditions indicated under Example 1f. In the elution profile arethe 6 peaks which correspond to the peptides 1-6 of Example 1 g,numbered continuously. Relative absorption units at a wavelength of 206nm are indicated on the Y axis, the time in minutes is indicated on theX axis.

FIG. 3 (A): Time course of level of mRNA for 135 kD DEAH-box protein innormal A20.2J cells and leflunomide-resistant A20R cells treated withA77 1726. The hybridization was carried out using the radiolabeled DNAprobe A20-5/6b, whose sequence contains the preserved regions of theDEAD box protein DEAH subfamily. Probe A20-5/6b was made by using theprimers A20-5 and A20-6b and A20R DNA as template DNA. The molecularweight marker used was the RNA length standard I from BoehringerMannheim. In the 1st track the A20R entire RNA is applied, in the 2ndtrack A20.2J entire RNA without treatment of the corresponding cellswith A77 1726, in the 3rd-6th track A20.2J entire RNA in each case withincubation of varying length of the corresponding cells with 5 μM A771726 (1 hour, 8 hours, 16 hours, 24 hours). 20 μg of entire RNA of eachbatch were applied.

FIG. 3(B): the same blot as under (A) has been hybridized with a β-actinsample as control.

FIG. 4 (A) Northern experiment for the expression of the 135 kD DEAH-boxprotein after removal of leflunomide in leflunomide-resistant A20Rcells. The control was the RNA of A20R cells which had been incubatedwith 100 μM leflunomide (track 1). Hybridization was carried out withthe DNA probe A20-5/6b. The tracks 2, 3, 4, 5, 6, 7 and 8 each contained15 μg of entire RNA from A20R cells which had been incubated withoutleflunomide over the periods 1, 2, 3, 4, 5, 14 days and 5 months.

FIG. 4(B): Control hybridization of the same blot with a β-actin sample.The blots are always shown with the appropriate quantitative assessment.

FIG. 5: Northern blot with about 2 μg of poly (A) RNA per track of eightdifferent human tissues. The tracks 1-8 contain, from left to right,tissue from the heart, brain, placenta, lungs, liver, skeletal muscle,kidney and pancreas. The RNA was separated electrophoretically on adenaturing 1.2% strength agarose gel, and then blotted on a positivelycharged nylon membrane, then fixed by UV crosslinking. Hybridization wascarried out with the A20-5/6b DNA probe. The appropriate quantitativeassessment is shown under the blot.

FIG. 6(A): Results of the initial sequencing and restriction mapping ofthe isolated positive clones. Clones 1 and 3 are nearly identical.Clones 1/3 to 4 overlap and have the hs1/hs2 sequence in the insert.Clones 1/3 and 2 have a common Sph I cutting site. cDNA 4 liescompletely in cDNA 2. Clone 5 differs from the other clones by the size(6.5 kb), the restriction cutting sites and the missing hs1/hs2 cDNA.

FIG. 6(B): Homology domains in the sequence of the cDNA. The homologydomains are framed and the distance in amino acids between the domainsis indicated. Nine DEAH box homology domains are shown. The domain NLShas homology to the "nuclear localization site" from the T antigen (SEQID NO:2 is shown in this figure.)

FIG. 7: (A) Primer construction (SEQ ID NOS 7-14, respectively) of asubregion of the 135 kDa protein from A20R, which is expressed to anincreased extent. The series of letters in each case characterize theamino acids in the single letter code (SEQ ID NOS 28-33, respectively;under this the nucleotide sequence is indicated. The amino acidsequences written in brackets are listed beginning with their C-terminalend and are derived from DNA sequences which are complementary to theprimer sequences given here. In each case the degenerate genetic code isgiven. As the third base of the codon is often not clear, in order ineach case to obtain the appropriate base for the corresponding aminoacid, a mixture of all possible bases is synthesized. N is theabbreviation for all four bases (G, A, T, C). I is the abbreviation forinosine, which enters into base pairing with purine and pyrimidinebases. R=A, G; Y=T, C; S=G, C. A20-2, A20-3, A20-4 and A20-5 aredegenerate primers situated upstream. A20-6a and A20-6b are primerssituated downstream. The average distance of the primers situatedupstream and downstream to one another is approximately 600 nucleotides.In the case of the primer A-20-6b indicated under 6, the 16th nucleotidewas inadvertently set equal to N, so that here in the correspondingcomplementary strand the coding is both for isoleucine (ATT, ATC, ATA)and for methionine (ATG). This fact did not affect the success of thePCR carried out, but in this way a methionine appears falsely as thesixth-last amino acid in the sequence as in FIG. 8 and not the correctisoleucine. (B) Primer derived from the human cDNA clone B 185;7=downstream primer; 8=upstream primer.

FIGS. 8a and 8b: Sequencing of the subregion of the 135 kD DEAH-boxprotein from leflunomide-resistant A20R cells. Below the base sequence(SEQ ID NO:15) (1-612) the corresponding amino acid sequence (SEQ IDNO:16) is given in the single letter code. Isoleucine and not methionineis correct as the sixth to last amino acid; for explanation see legendto the figure for FIG. 7. The DNA fragment shown was used as anA20-5/-6b probe for the hybridization experiments.

FIG. 9a-9i: Sequence of the coding region of the entire human cDNA (4272bp total length). From the position of the homologies to the mousesequence, it followed that the first reading frame was correct. Thecoding sequences lie between positions 148 and 3831 (SEQ ID NO:17) andyield a sequence of 1227 amino acids (SEQ ID NO:18). (*=stop)

FIG. 10: Similarities of the gene for the human 135 kD DEAH-box proteinwith other DEAH-box proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the identification andmolecular-biological and biochemical characterization of novel proteinswhich show structural similarity with other known helicases. It isdemonstrated that such proteins bind ATP and nucleic acid and possesshelicase and ATPase activities. This invention further relates toprocesses for preparation of such proteins and their use inpharmacologically relevant test systems and therapeutic applications.

In order to identify potential intracellular sites of action ofleflunomide, a leflunomide-resistant cell line was developed (see alsoExample 1). This resistance was induced against A77 1726 in the highlyproliferative cell line A20.2J (murine B-cell lymphoma). Theconcentration of A77 1726 was increased stepwise in a serum-free culturesystem, which finally led to the establishment of a stable subline namedA20R. The A20R cell line tolerates 30-40 times higher leflunomideconcentrations than the original cell line A20.2J (ED₅₀ 130 μM comparedwith 4 μM).

Surprisingly, it has now been found that by means of such a treatment ofa leucocyte cell line with rising, but nontoxic, doses of theantiproliferative active compound leflunomide, the expression of ahitherto unknown 135 kD DEAH-box protein is induced. This protein is a"DEAH-box leflunomide-resistant protein" (defined below). The helicaseand ATPase activities of this protein are assessed using techniques thatare well known to the skilled artisan. The ability of this protein tobind RNA or otherwise affect RNA homeostasis is also assessed usingmethods well known to the skilled artisan. Stimulation of RNA helicaseactivity in leflunomide-resistant cells would enable the cells toproliferate, probably by means of more efficient utilization of existingtranscripts.

As used in this specification a "DEAH-box leflunomide-resistant protein"is used to mean a protein that has the ability to bind nucleic acid andATP, and has ATPase and helicase activities, wherein the level ofexpression of said protein is significantly higher in aleflunomide-resistant cell than in a non-leflunomide-resistant cell,with the proviso that such a "DEAH-box leflunomide-resistant protein" isnot a protein that was publicly known prior to Dec. 4, 1995.

As used in this specification, "publicly known" means known or used byothers in the United States, or patented or described in a printedpublication in the United States or a foreign country, or in public use,or on sale.

As stated above, a DEAH-box leflunomide-resistant cells is expressed ata significantly higher level in a cell that tolerates high levels ofleflunomide (leflunomide-resistant cell), when compared with the proteinexpression level in a corresponding cell line that does not toleratehigh levels of leflunomide. For example, the ED₅₀ for leflunomide inmurine A20.2J cells is 4μ, whereas the ED₅₀ for murine A20.2J cells thatare resistant to high levels of leflunomide is 130μ. (See Example 1below.) As used in this specification, a "leflunomide resistant" cell isone that will tolerate a high level of leflunomide. Conversely,"non-leflunomide-resistant cells" will not tolerate these high levels ofleflunomide. Tolerance to leflunomide is indicated by the ability toproliferate in the presence of high levels of leflunomide. As used inthis specification, a "high level of leflunomide" is used to meanconcentrations in the range of about 100 μM to about 150 μM. As used inthis specification, a first cell that expresses a "significantly higherlevel" of a particular protein than a second cell expresses about 20times to about 100 times as much of the particular protein as the secondcell, as expressed per milligram of total cell protein.

The invention also includes a protein comprising the amino acid sequenceshown in FIG. 8 (SEQUENCE ID NO. 16) and the amino acid sequence shownin FIG. 9 (SEQUENCE ID NO. 18). Other embodiments of the inventioninclude a "fragment" of an amino acid sequence as set forth in FIG. 8(SEQ. ID NO. 1 6) and a "fragment" of anamino acid sequence as set forthin FIG. 9 (SEQ. ID NO. 18). As used in this specification, a "fragment"of the FIG. 8 or FIG. 9 sequence is all or part of the FIG. 8 or FIG. 9sequence, with the proviso that such a fragment is not a fragment thatwas publicly known prior to Dec. 4, 1995.

The invention also encompasses DEAH proteins with the ability to bindnucleic acid and with helicase and ATPase activities that have beenisolated from mammalian cell lines, including a human cell line or aderivative of the murine cell line A20.2J.

In another embodiment, the invention provides a protein that has amolecular weight of 135 Kd and that retains the "essential identifyingcharacteristics" of a protein according to the invention--having theability to bind nucleic acid and ATP, and having ATPase and helicaseactivities. In yet another embodiment, the invention provides such aprotein having a molecular weight of about 135 Kd. The skilled artisanwill recognize that proteins having other molecular weights are alsoencompassed by the invention.

In yet another embodiment, the invention relates to an isolated DEAH-boxprotein having or more of the following characteristics: (1) the firsthomology domain (ATPase A, Domain I) is located more than 650 aminoacids from the N-terminus; (2) there are Serine/Arginine (SR) domains inthe N-terminus (3) domain IV has the sequence FMP; (4) the distancebetween domains IV and V is 74 amino acids or less; (5) domain VI hasthe sequence QRSGRXGR (SEQ ID NO:25). Homology domains are shown in FIG.6B.

The present invention also relates to preparing in a manner which isconventional and known from the literature monoclonal and polyclonalantibodies against the entire protein, parts of the protein and peptidesequences obtained by proteolytic degradation or peptide synthesis. See,for example, Coligan, et al. (eds.) CURRENT PROTOCOLS IN IMMUNOLOGY, pp.2.0.3-2.11.8 (John Wiley & Sons, 1992). The present invention alsorelates to purification processes for the functional protein. Theinvention further relates to the structural and functionalcharacterization of the protein according to the invention. Suchcharacterization may be carried out using both molecular biological andbiochemical techniques that are well known to the skilled artisan.

The present invention also relates to methods for identifying, isolatingand cloning a gene or gene subsequences encoding such proteins accordingto methods which are conventional and known from the literature. Theinvention also relates to methods for expressing such genes or genesubsequences in suitable expression systems.

A further embodiment of the invention is an isolated DNA sequence whichencodes a protein according to the invention. The invention includes theDNA sequence shown in FIG. 8 (SEQUENCE ID NO. 15) and the DNA sequenceshown in FIG. 9 (SEQUENCE ID NO. 17).

A further embodiment of the invention is a "homolog" of the DNA sequencein FIG. 8 (SEQ. ID NO. 15) and a homolog of the DNA sequence shown inFIG. 9 (SEQUENCE ID NO. 17). As used in this specification, a "homolog"of the sequence of FIG. 8 (SEQ. ID NO. 15) is a nucleotide sequencewhich under stringent conditions, hybridizes to the DNA sequence as inFIG. 8 or to a subsequence of the sequence in FIG. 8 (SEQ. ID NO. 17),with the proviso that said sequence is not a DNA sequence encoding amember of the DEAD-box protein family that was publicly known prior toDec. 4, 1995. Similarly, a "homolog" of the sequence of FIG. 9 is a DNAsequence that hybridizes under stringent conditions to the DNA sequenceas in FIG. 9 or to a subsequence of the sequence in FIG. 9, with theproviso that said sequence is not a DNA sequence encoding a member ofthe DEAD-box protein family that was publicly known prior to Dec. 4,1995.

Stringency, as used in this specification, means the condition withregard to temperature, ionic strength and the presence of certainorganic solvents, under which nucleic acid hybridizations are carriedout. As used in this specification, "stringent conditions" forhybridization is used to designate the following types of stringentwashes: (1) 68° C., or about 68° C. using ExpressHyb solution (Clontech,Heidelberg, Germany)(radioactive label): (2) 40-600° C. in DigEasyHybsolution (Boehringer Manheim)(nonradioactive label). In alternativeembodiments, "stringent conditions" means hybridization at about 43° C.in DigEasyHyb solution for identification of DNA/DNA hybrids orhybridization at about 50° C. in DigEasyHyb solution for identificationof DNA/RNA (including mRNA) hybrids. In another alternative embodiment,"stringent conditions" includes hybridization in 0.1×SSC and 0.1% SDS at40-60° C. The skilled artisan will recognize that the precise stringenthybridization parameters may be optimized, depending on experimentalconditions.

Thus, in another embodiment, the present invention relates to DNAsequences that will hybridize to the DNA sequence of FIG. 8, FIG. 9, ora subsequence of either sequence under the following stringentconditions: ExpressHyb solution (Clontech, Heidelberg, Germany) at 68°C. or at about 68° C. Further washing techniques are set forth inExample 6.

A further embodiment of the invention is a DNA which, on account of thedegeneracy of the genetic code, is different from the DNA sequences ofthe invention (as illustrated in FIGS. 8 and 9), but which expresses aDEAH box protein that binds nucleic acid and ATP, and which has helicaseand ATPase activities. The skilled artisan will recognize thatconservative nucleotide changes may be made that will encode the sameamino acid sequence of the 135 kD DEAH-box proteins described herein. Inaddition, the skilled artisan will also recognize that the nucleotidechanges in a DNA sequence according to the invention can be made toeffect conservative amino acid substitutions. These changes may be madeso that such proteins made will retain the "essential identifyingcharacteristics" of a protein according to the invention--having theability to bind nucleic acid and ATP, and having ATPase and helicaseactivities.

One of skill in the art will recognize that such DNA sequences may bemade using many techniques that are well-known in the art, such assynthetic oligonucleotide synthesis, site-directed mutagenesis. Inaddition, DNA sequences according to the invention can be identified byusing all or part of a nucleotide sequence disclosed in thisspecification as a probe to screen genomic or cDNA libraries. Suchtechniques are well-known to the skilled artisan. For example, asuitable subregion of the human gene is nucleotide 1594 to nucleotide2205. In addition, the entire sequence of a human or murine DEAH-boxprotein according to the invention can also be used as a probe.

The invention includes a DNA sequence that encodes a protein that hasthe ability to bind nucleic acid and ATP, and has ATPase and helicaseactivities, wherein the level of expression of said protein issignificantly higher in a leflunomide-resistant cell than in anon-leflunomide-resistant cell.

In another embodiment, the invention provides a DNA sequence thatencodes a protein that has a molecular weight of 135 kD and that retainsthe "essential identifying characteristics" of a protein according tothe invention--having the ability to bind nucleic acid and ATP, andhaving ATPase and helicase activities. In yet another embodiment, theinvention provides such a protein having a molecular weight of about 135kD. The skilled artisan will recognize that proteins having othermolecular weights are also encompassed by the invention.

In yet another embodiment, the invention relates to a DNA sequence thatencodes a protein having or more of the following characteristics: (1)the first homology domain (ATPase A, Domain I) is located more than 650amino acids from the N-terminus; (2) there are Serine/Arginine (SR)domains in the N-terminus (3) domain IV has the sequence FMP; (4) thedistance between domains IV and V is 74 amino acids or less; (5) domainVI has the sequence QRSGRXGR (SEQ ID NO:25). Homology domains are shownin FIG. 6B.

In addition, the invention relates to a vector which comprises a DNAsequence encoding a protein according to the invention and which issuitable for the expression of said protein in a suitable host cell. Anexpression vector for a suitable host cell is a vector which in theappropriate host cell is capable of heterologous gene expression and ofreplication, constitutively or after induction by means of customarymethods. Suitable vectors include, but are not limited to, PSEAP, PCMV,pSV, pTK, pcDNAI (Clontech, Heidelberg, Germany). In one embodiment,those vectors that carry out gene expression and replication with highefficiency are used.

Another embodiment of the invention is such a host cell containing avector according to the invention. Suitable host cells include, but arenot limited to, Jurkat T-cells, Raji B-cells, A20 cells, Hela cells,insect cells for Bacculo virus expression systems.

In addition, the invention relates to an "antisense" expression vector.Such an expression vector contains a DNA sequence according to theinvention, which is inserted in reverse orientation in the expressionvector. Thus, the skilled artisan will recognize that the mRNAcorresponding to the DNA in the antisense vector will hybridize with anmRNA corresponding to the DNA in the "sense" orientation vector. Anantisense expression vector is a vector which expresses a desiredantisense RNA in an appropriate host cell, either constitutively orafter induction by means of customary methods.

In another embodiment, the invention provides processes for thepreparation of the a protein according to the invention by expression ofthe protein by means of the vectors and host cells mentioned andsubsequent isolation of the protein using customary methods that arewell known to the skilled artisan. For example, affinity purification,HPLC, and FPLC can be used.

By making available this protein and related RNA helicases, novelanticarcinogenic, anti-atherosclerotic, immunosuppressive,antiinflammatory, antiviral, antifungal and antibacterial activesubstances are identified. These are urgently needed for the efficienttherapy of a whole host of diseases, such as, for example, Alzheimer'sdisease, cancer, rheumatism, arthrosis, atherosclerosis, osteoporosis,acute and chronic infectious diseases, autoimmune disorders, diabetesand organ transplant rejection. The skilled artisan will recognize thatpreparation of pharmaceutical preparations of such substances are wellknown in the art.

Thus, the invention furthermore relates to the use of a proteinaccording to the invention in a test or assay system for finding novelor identifying already known substances which have anticarcinogenic,anti-atherosclerotic, immunosuppressive, antiinflammatory, antiviral,antifungal or antibacterial action. In one embodiment, a proteinaccording to the invention is prepared using genetic engineering, orrecombinant DNA, methods. Such an assay system can be designed such thata protein according to the invention is crystallized and itsthree-dimensional structure is elucidated using customary methods. See,for example, A. McPherson, PREPARATION AND ANALYSIS OF PROTEIN CRYSTALS(John Wiley & Sons, 1982) and Ducruix, et al., eds. CRYSTALLIZATION OFNUCLEIC ACIDS AND PROTEINS (Oxford University Press, Oxford, 1992).

Using customary methods of "molecular modeling," substances areidentified or developed which react with a protein according to theinvention. Thus, a substance is identified that affects the ability ofsuch a protein to bind ATP or other substrates, such as DNA, RNA andRNA/protein complexes. Substances which interfere with the proteinsubstrate binding site(s) or at a site which affects such functionalepitopes are identified. Molecular modeling techniques are known in theart. See, for example, Fruehbeis, et al., Int. Ed. Engl. 26:403 (1987);Perun, etal., eds. COMPUTER-AIDED DRUG DESIGN (Marcel Dekker, Inc.: NewYork, 1989)); van de Waterbeemd, ADVANCED COMPUTER-ASSISTED TECHNIQUESIN DRUG DISCOVERY (Verlagsgesellschaft, Weinheim, 1994); and Blundell,Nature, 384:23 (1996), all of which are hereby incorporated byreference.

Substances are also tested for their ability to modulate the enzymaticactivities of a protein according to the invention. As used in thisspecification, a substance that "modulates" an enzyme activity causes achange in the enzyme activity when compared to the enzyme activity asmeasured in the absence of the test substance. For example, a substancemay partially or totally inhibit the enzyme activity. The test for RNAhelicase activity is carried out by methods known to the person skilledin the art. For example, synthetic oligoribonucleotides can beimmobilized on a matrix and hybridized with complementary, labeledoligoribonucleotides. The hybridized oligos are then contacted with aprotein according to the invention, which releases a certain, measurableamount of the labeled, non-matrix-immobilized oligoribonucleotides, dueto the helicase activity of the protein. The effect of the presence orabsence of potential modulators on the helicase is tested.Alternatively, the procedure described by Jaramillo, et al. Mol. Cell.Biol. 11:5992 (1991). Briefly, duplex RNA substrate (labeled with ³² p)is mixed with helicase protein in a buffered solution and the reactionis terminated using glycerol/SDS/EDTA/bromphenol blue. This reactionmixture is applied to an SDS gel (8%) and the gel is run using standardprocedures. Unwinding efficiency is defined as the ratio of unwoundmonomer RNA relative to duplex RNA. Other assay procedures are wellknown in the art. See, for example, Rozen, et al. Mol. Cell. Biol.10:1134 (1990) and Pause, et al. EBMO J. 11:2643 (1992), which arehereby incorporated by reference. Such assays can also be carried out onmicrotiter plates, by which means a large number of modulators can betested for their action with high efficiency.

Further assays for modulators of a protein according to the invention isATPase or splicing tests in which the effect of modulators is tested onthe ATPase or splicing properties of a protein according to theinvention.

The test for ATPase activity is carried out using procedures that arewell known in the art. For example, the procedure described by Pause, etal. EBMO J. 11:2643 (1992) is used. Briefly, a protein according to theinvention is incubated at 37° C. in a buffered solution containing ³²P-labeled ATP. The reaction is stopped by the consecutive addition ofthe following reagents at 4° C.: 20 mM silicotungstate, 1 mM potassiumphosphate, 5% ammonium molybdate/4M sulphuric acid, 2.5% trichloroaceticacid/50% acetone, 50% isobutyl alcohol/50% benzene. The mixture isvortexed and centrifuged. The upper phase is assayed for radioactivityto determine ATP hydrolysis.

A protein according to the invention is also tested for its RNA splicingactivity. A splicing assay is carried out using procedures that are wellknown in the art. For example, the procedure of Tarn, et al., Cell84:801 (1996) or the procedure of Xu, et al. Nature, 381:709 (1996),which are hereby incorporated by reference, may be used.

In another embodiment of the invention, modulators of a proteinaccording to the invention are identified using the following procedure.Cells that are not resistant to high levels of leflunomide aretransformed with a vector containing a gene encoding a protein accordingto the invention. These transformed cells are grown in the presence ofleflunomide. These cells tolerate leflunomide because they express aprotein according to the invention. Various compounds are tested fortheir ability to inhibit the growth of the transformed cells. Thosecompounds that cause the cells to die or decrease cell proliferation arelikely inhibiting the activity of the protein according to theinvention. Thus, such substances are further characterized for theirability to modulate the activities of a protein according to theinvention--ability to bind ATP and nucleic acid and having helicase andATPase activities.

Accordingly, other embodiments of the invention include therapeuticmethods for treatment of various disorders comprising administering atherapeutically effective amount of a substance (identified using theabove-described methods) to a patient in need of such treatment.

In another embodiment of the invention, a protein according to theinvention is used to isolate RNAs binding specifically to such aprotein. The oligoribonucleotide sequence(s) of RNAs binding to aprotein according to the invention can then be determined. To isolatesuch RNAs, a protein according to the invention (or a fragment thereof)is coupled to a matrix. The affinity matrix prepared in this way is usedto concentrate RNAs, which specifically bind to the coupled protein orparts thereof, from RNA mixtures. Binding can be measured by standardmethods like Electrophoretic Mobility Shift Assay (EMSA) or ELISA, whichare well known in the art. The following assays may also be used:Scintillation Proximity Assay (SPA, Amersham International, LittleChalfont, Buckinghamshire, England) and BlAcore (Biomolecule InteractionAnalysis, Pharmacia, Upsala Sweden).

The RNA fragments obtained from the affinity matrix are then amplifiedusing various PCR primers (or linkers) and the amplified fragments arethen sequenced using techniques well known to the skilled artisan. PCRprimers are selected using an oligo dT primer (3' end) in combinationwith a degenerate primer (5' end). Alternatively, the RapidAmplification of cDNA Ends protocol can be used. See Innis, et al., eds.PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS. Academic Press: SanDiego, 1990.

Yet another embodiment of the invention is the use of a DNA sequenceaccording to the invention as a selection marker. Such a DNA sequencecan be used as a marker when it is inserted into a vector. In thisembodiment of the invention, use is made of the observation that in aSouthern blot analysis of genomic DNA of the A20R cells in comparisonwith genomic DNA of the A20.2J cells the mRNA corresponding to the genewhich encodes a 135 kD DEAH-box protein is increased, or amplified.Thus, a vector comprising a DNA sequence according to the invention canbe inserted into cells that are not naturally resistant to leflunomide.Cells containing the foreign, or heterologous, DNA containing the geneencoding a protein according to the invention will grow in the presenceof leflunomide, and hence be selected. In yet another embodiment of theinvention, cells can be transformed with a vector comprising a geneencoding a protein that is amplified in cells that are resistant toleflunomide analogs.

The gene amplification by leflunomide or leflunomide analogs observed asexemplified by this gene is used in the selection of cells and in genetherapy. In some gene therapy applications, it may be advantageous totreat a patient with a vector encoding a protein that will substitutefor a defective protein that is produced by the patient in need of genetherapy. As noted above, a variety of diseases are associated withoverexpression of helicase activity. Thus, overexpression of a proteinaccording to the invention is determined. The protein and mRNA levelsfor such a protein are compared in normal individuals and thoseindividuals with a disease, such as a hereditary disease, an autoimmunedisease, or cancer. In those diseases having amplified expression of aprotein according to the invention, such amplification is used as amarker for the presence of disease. In some instances, the amplificationis used as a marker for those individuals at risk for developing aparticular disease.

The invention will now be illustrated in greater detail with the aid ofthe figures and examples, without being restricted thereto.

EXAMPLE 1 Preparation of the Leflunomide-Resistant Cells

Medium:

The culturing of the starting line, the breeding of the resistantsubline A20R and the proliferation tests for checking thecross-resistance of the A20R cells were performed in a self-prepared,serum-free medium. Dry medium for 10 liters of Iscove medium (Biochrom,Berlin) was dissolved in 10 liters of double-distilled water.

18.95 9 of NaCl

11.43 g of NaHCO₃

700 mg of KCl

10 ml of 35% strength NaOH solution

0.5 ml of 1 molar mercaptoethanol solution

were then added to the solution and the medium was sterile-filtered (allsubstances from Riedel de Haen).

Before use

32 mg of human holo-transferrin

1 g of bovine albumin

1.5 ml of lipids

(all substances from Sigma) were added to 1 liter of Iscove medium.

Description of the starting line A20.2J:

A20.2J is a subline of the mouse B-cell lymphoma A20 (ATCC TIB-208) anddescribed as a fusion line in the ATCC for the cell line LS 102.9 (ATCCHB-97). The cells were distinguished by high proliferation (doublingtime about 10 hours) and a high sensitivity (50% inhibition of theproliferation of the cells at 2 μM substance) to A77 1726 (the mainmetabolite of leflunomide). The cells were easy to culture as anonadherent-growing cell line.

Description of resistance breeding:

A20.2J cells were initially cultured for 5 days in Iscove medium with 1μM A77 1726 (concentration below the 50% inhibition of proliferation)and the cell growth and the vitality of the cells were checked. Every2nd or 3rd day, the cells were passaged in fresh medium to which thesame concentration of A77 1726 was added. After culturing for 5 days,growth of the cells and a low dying-off rate (maximum 30% dead cells)was detectable, so the concentration of A77 1726 was increased stepwise.If the proliferation of the cells stagnated, the concentration of thelast passage was used. After culturing for one year, a stable, resistantsubline A20R was established which, in the presence of 100 μM A77 1726,showed constant proliferation and no differences morphologically to thestarting line A20.2J.

Detection of proliferation:

5×10⁵ cells were incubated in 5 ml of Iscove medium in 6-well plates(Greiner) for 48 hours at 37° C. and 10% CO₂.

One well was set up as a positive reference value:

for A20.2J:cells in Iscove medium

for A20R:cells in Iscove medium+100 μM A77 1726

Test substances in various concentrations were pipetted into the cellsin the remaining wells. After the incubation time, the cells wereresuspended in the well, and 100 μl of cell suspension were taken anddiluted in 1% strength Eosin solution (1 g of Eosin yellowish fromRiedel de Haen dissolved in 100 ml of sterile isotonic saline solution).The cells were counted in a Neubauer counting chamber and the fractionof dead cells (stained by Eosin) determined. The substance-inducedalteration of proliferation was calculated relative to the respectivepositive control.

Test 2:

4×10³ cells were pipetted into a volume of 100 μl of Iscove medium in96-well round-bottom microtiter plates (Nunc). Test substances wereapplied in twice the concentration starting from the desired testconcentration and 100 μl of this solution was pipetted into the cells.The plates were incubated for 48 hours at 37° C. and 10% CO₂. Theproliferation was determined by radiolabeling the DNA of dividing cells.To do this, after the incubation time 25 μl of ³ H-thymidine (10 μCi/ml;specific activity 29 Ci/mmol; Amersham) was added to each well and themixture was incubated for a further 16 hours. To evaluate the test, theplates were harvested on glass fiber filters (Pharmacia) by means of acell harvester (Skatron), unincorporated ³ H-thymidine being collectedin separate waste flasks, and only cellular, DNA-bound radioactivitybeing measured. The filters were heat-sealed in plastic bags and afteraddition of 10 ml of scintillator (Pharmacia) sealed in countingcassettes for measurement. Measurement was carried out in a beta-counter(beta-plate system 1206 from Wallac). As indicated under Test 1, thealteration in proliferation of the test substances was calculatedagainst the respective positive controls.

EXAMPLE 2 Test for the Resistance of the A20R Cells

1. Cross-resistance to antiproliferative substances known from theliterature:

Antiproliferative substances known from the literature were tested atdifferent concentrations (as described in proliferation test 2) fortheir antiproliferative properties on A20R cells and A20.2J. In thefollowing table, the calculated inhibition of a concentration of thesesubstances on both cells lines is shown. The resistance of the A20Rcells to antiproliferative substances is compared with the starting lineA20.2J.

    ______________________________________                                                            % inhibition of                                                                            % inhibition of                              Test substances     A20.2J       A2OR                                         ______________________________________                                        Methotrexate                                                                             (0.15 μM)                                                                           75.9         65.2                                         Cisplatin  (10 μM)                                                                             44.7         91.1                                         Cyclosporin A                                                                            (0.25 μM)                                                                           69.9         77.5                                         Mycophenolic acid                                                                        (0.15 μM)                                                                           89.8         76.8                                         ______________________________________                                    

2. Cross-resistance to structurally related substances similar to A771726.

As no general resistance of the A20R cells to antiproliferativesubstances was present (Example 2(1)), it was determined whetherstructurally related analogs of A77 1726 have the sameproliferation-inhibiting properties on A20R cells as on A20.2J cells.The investigation was carried out by means of proliferation test 1. Inthe table which follows, comparative IC₅₀ values (the concentration of asubstance which inhibits the proliferation of the cells by 50%) areshown.

    ______________________________________                                                       IC.sub.50 value of                                                                      IC.sub.50 value of                                   Test substances                                                                              A20.2J    A20R                                                 ______________________________________                                        A77 1726       2-3 μM 130 μM                                            X92 0715        8 μM  120 μM                                            X91 0279       10 μM  120 μM                                            X91 0325       10 μM   75 μM                                            ______________________________________                                    

A20R cells show a gradually decreasing cross-resistance to structurallyrelated A77 1726B analogs, which suggests a structure-specificresistance.

3. Cross-resistance of the A20R cells to brequinar:

Earlier investigations on the mechanism of action of leflunomide pointedto parallels with brequinar (Dupont-Merck). For this reason, brequinarwas additionally included in the investigations on the cross-resistanceof A20R. Brequinar is not a structural analog of leflunomide.

The IC₅₀ values of the A20.2J and A20R cells to the brequinar sodiumsalt were determined with the aid of proliferation test 1.

    ______________________________________                                                  IC.sub.50 value of A20.2J                                                                IC.sub.50 value of A20R                                  ______________________________________                                        Brequinar Na.sup.+                                                                        0.2 μM    50-75 μM                                          salt                                                                          ______________________________________                                    

A20R cells show with respect to their growth behavior a cross-resistanceto analogs of A77 1726 and brequinar, a substance which inhibits DHODH.

EXAMPLE 3 Investigation of A20R Cells for MDR Proteins

Gel electrophoresic separations of the cellular proteins of the A20.2Jand A20R cells showed that a protein having a molecular weight of above135 kDa (determined using protein calibration markers) was overexpressedin the resistant line (see also FIG. 1). This example evaluates whetherthe 135 kDa protein is a MDR (multi-drug resistance) protein.

MDR (multi-drug resistance) is defined as a resistance of the cells tostructurally unrelated antineoplastic substances. Tumor cells react byoverexpression of a plasma membrane glycoprotein which can pump outATP-dependent cytotoxic substances from the cells. By overexpression ofthese MDR proteins (135-180 KD), the cells survive even in relativelyhigh concentrations of antiproliferative substances.

The function of MDR proteins as secretory pumps can be inhibited bycalcium channel blockers, which leads to an accumulation of thesubstance in the cell. Calcium channel blockers known from theliterature and also MDR-associated substances were therefore added toboth cell lines in order to check whether the resistant lineoverexpressed MDR proteins. The calcium channel blocker used wasverapamil, the MDR substrates used were daunorubicin and doxorubicin.The results are shown below in tabular form as % inhibitlion ofproliferation and were determined with the aid of Test 2.

    ______________________________________                                        Verapamil(nM)                                                                            A.20.2J* A20R*     A20.2.J**                                                                            A20R**                                   ______________________________________                                        0          10.7%    6.8%      2.7%   9.1%                                     100        33.4%    20.7%     19.9%  24.9%                                    200        49.6%    31.7%     30.4%  48.7%                                    400        54.0%    42.4%     40.4%  47.3%                                    ______________________________________                                         *Addition of 300 nM daunorubicin                                              **Addition of 300 nM doxorubicin                                         

Both cell lines are inhibited by the two substances to the same extent.Thus, the resistant A20R cells do not show a higher acceptance due toincreased MDR expression. Thus, the higher tolerance of the A20R cels isnot due to an induction of MDR proteins, but is due to some otherfactor. The same test mixture was chosen in order to check whether A771726 is an MDR-transported molecule. Numbers are % inhibition ofproliferation.

    ______________________________________                                        Verapamil                 A20R +                                              (nM)      A20.2J + 1.6 μM A77 1726                                                                   62.5 μM A77 1726                                 ______________________________________                                        0         16.4%           10.3%                                               100       14.3%           6.4%                                                200       12.5%           9.9%                                                400       7.9%            13.9%                                               ______________________________________                                    

In the case of these cell lines, it was determined that A77 1726 is nottransported by MDR proteins. Thus, the leflunomide resistance of theA20.R cells is not caused by the action of MDR proteins, transportingthe leflunomide out of the cells.

EXAMPLE 4 Micropreparative Purification of a 135 kD Protein

a.) Sample preparation for protein determination

a.1) Protein concentration was determination using the methods describedby Popov, et al., Acta Biol. Med. Germ. 34, pp.1441-1461.

The principle of the method is that dilute protein solutions areprecipitated as colored pellet using Napthol Blue/Black/methanol/aceticacid, washed, taken up in 0.1 M NaOH and the extinction is measured at620 nm.

The protein content is calculated by means of a calibration curve usingBSA solutions (BSA=bovine serum albumin).

This method for protein determination is not affected by detergents(SDS, Nonidet, etc.) and the presence of β-mercaptoethanol does notinterfere with the method either. Original Eppendorf vessels should beused, as the adhesion of the pellets to the plastic surface is strongand protein losses due to dissolution of the pellets on pouring off thewash solutions are avoided.

The following solutions are needed:

    ______________________________________                                        "Popov 1 solution":                                                           stir 0.65 g of Naphthol Blue/Black                                            + 50 ml of Popov 2 for at least 1 h, can only be kept for one                 week.                                                                         "Popov 2 solution":                                                           50 ml of glacial acetic acid                                                  + 450 ml of methanol                                                          "Popov 3 solution":                                                           4 ml of Popov 1                                                               + 36 ml of Popov 2, then filter                                               ______________________________________                                    

Plotting the calibration curve:

Preparation of the BSA solution: bovine albumin, from Sigma, 98 to 99%purity is prepared in a concentration of 1 mg/ml in 5% strength SDSsolution. A relatively large amount of solution is prepared, which isstored in 1 ml portions at -25° C. A 1 ml portion is thawed and thenvigorously shaken for 10 minutes at 95° C. in a thermomixer (Eppendorfthermomixer 5436).

    __________________________________________________________________________    After cooling, the following dilutions are performed:                         __________________________________________________________________________    10  μl of BSA solution                                                                  +990                                                                              μl of 5% strength SDS solution                                                           0.010                                                                            mg of BSA/ml                                25  μl of BSA solution                                                                  +975                                                                              μl of 5% strength SDS solution                                                           0.025                                                                            mg of BSA/ml                                50  μl of BSA solution                                                                  +950                                                                              μl of 5% strength SDS solution                                                           0.050                                                                            mg of BSA/ml                                75  μl of BSA solution                                                                  +925                                                                              μl of 5% strength SDS solution                                                           0.075                                                                            mg of BSA/ml                                100 μl of BSA solution                                                                  +900                                                                              μl of 5% strength SDS solution                                                           0.100                                                                            mg of BSA/ml                                150 μl of BSA solution                                                                  +850                                                                              μl of 5% strength SDS solution                                                           0.150                                                                            mg of BSA/ml                                200 μl of BSA solution                                                                  +800                                                                              μl of 5% strength SDS solution                                                           0.200                                                                            mg of BSA/ml                                without                                                                           μl of BSA solution                                                                  +1000                                                                             μl of 5% strength SDS solution                                                           blank value                                    __________________________________________________________________________

200 μl of all 8 solutions are in each case taken twice (duplicatedetermination), mixed with 600 μl of "Popov 3", then mixed brieflyvigorously (vortex).

Centrifuge for 5 minutes at 14000 rpm in a bench-top centrifuge(Eppendorf), the supernatant is discarded. The pellet is then washed 3times with 750 μl of "Popov 2" each time and centrifuged off. After thelast washing operation, the pellet is taken up in 1 ml of 0.1 M NaOH andthe extinction is measured in a plastic cuvette (d=1 cm) against theblank value at 620 nm (spectrophotometer from Kontron).

    ______________________________________                                        Example of a series of measurements:                                          Concentration BSA (mg/ml) Extinction at 620 nm                                ______________________________________                                                0     0                                                                       0.010 0.0459                                                                  0.025 0.1154                                                                  0.050 0.2442                                                                  0.075 0.4025                                                                  0.100 0.4964                                                                  0.150 0.6856                                                                  0.200 0.9534                                                          ______________________________________                                    

The correlation coefficient in the evaluation: proteinconcentration/extinction is, according to experience, 0.995-0.999 (inthis example 0.998)

a.2) Sample preparation/protein determination of the A 20 cells:

10⁷ A20 cells (the term A20 cells means both A20.2J and A20R cells),present in 1 ml of PBS buffer, are centrifuged for 5 to 10 seconds at10⁴ rpm in the bench-top centrifuge (Eppendorf model 5415 C). Thesupernatant is discarded, the pellet is mixed with 1 ml of 5% SDSsolution, and the mixture is sucked up with a pipette several times andthus homogenized and vigorously shaken for 10 minutes at about 95° C. inthe thermomixer and then cooled. Of this solution: 20 μl are mixed with980 μl of 5% SDS solution (50-fold dilution) and 50 μl are mixed with950 μl of 5% SDS solution (20-fold dilution) and the mixture isvigorously shaken for 10 minutes at 95° C. in the thermomixer andcooled. 200 μl of each solution are then taken twice for duplicatedeterminations, mixed with 600,μl of "Popov 3" and thus additionallytreated as described above for BSA. Evaluation is carried out with theaid of the calibration curve already described.

    ______________________________________                                        Measurements obtained:                                                                               →Protein conc. × dilution                                        factor                                                 Dilution Extinction at 620 nm                                                                        (mg/ml)                                                ______________________________________                                        50-fold  0.0972        0.915                                                  20-fold  0.1800        0.720                                                  ______________________________________                                         Result: A20 cells contain about 800 μg of protein/10.sup.7 cells.     

b.) Sample preparation for SDS-PAGE

10⁷ A20 cells, present in 1 ml of PBS buffer, are centrifuged for 5 to10 seconds at 10⁴ rpm in the bench-top centrifuge (Eppendorf model 5415C).

b. 1) Direct lysis

The supernatant is discarded, the pellet is mixed with 400 μl of samplebuffer and homogenized by sucking up several times with the pipette, andthe mixture is vigorously shaken (vortex shaker) and agitated for 5 to10 minutes at 95° C. in the abovementioned thermoshaker or water bath.The protein concentration of this highly viscous solution is about 2mg/ml. For a Coomassie-stained gel, 40 to 50 μl/sample bag of thissolution, corresponding to 80 to 100 μg of protein, are needed. ForAg-stained gels, the solution described is then additionally diluted1:20, 40 to 50 μl thus correspond to a protein concentration of 4 to 5μg/sample pocket.

Composition of the sample buffer:

    ______________________________________                                        Millipore H.sub.2 O                                                                              2.7 ml                                                     Glycerol, 98% strength                                                                           10.0 ml                                                    0.25M Tris/1M glycine                                                                            9.0 ml                                                     25% SDS soln.      6.8 ml                                                     0.1% Bromophenol Blue soln.                                                                      2.5 ml                                                     2-mercaptoethanol  4.0 ml                                                     ______________________________________                                    

b.2) Freezing of the cells and subsequent lysis

The cell pellet was immediately immersed in liquid nitrogen for about 1minute in the closed Eppendorf vessel and stored at -80° C. On lyzingthe sample buffer is added directly to the intensely cooled cell pellet.

c.) SDS-PAGE

Various polyacrylamide gels were used (10%, 12%, 4 to 22.5% PAA). Bestresults with respect to band sharpnesses were obtained using gradientgels whose PAA content was 4 to 10%. The techniques/solutions needed forthis are described below:

Separating gel

Composition of the gel solutions for gradient gel 4 to 10% AA for a gel(about 24 ml):

    ______________________________________                                                       4% AA                                                          Component      soln.       10% AA soln.                                       ______________________________________                                        H.sub.2 O      7      ml       --                                             Glycerol       --              6.1   g                                        Stock soln. 1  1.6    ml       4     ml                                       3M Tris, pH 8.8                                                                              3      ml       3     ml                                       10% APS        80     μl    40    μl                                    10% SDS        120    μl    120   μl                                    TEMED          10     μl    10    μl                                    ______________________________________                                    

Stock solution 1: 30% acrylamide/0.5% N,N'-methylenebisacrylamide

crosslinking: 1.7%

APS: ammonium persulfate

Collecting gel

Composition of the gel solution with 3.8% AA for two gels (about 10.5ml)

    ______________________________________                                        Component                                                                     ______________________________________                                        H.sub.2 O             3.7    ml                                               Stock soln. 2         4.0    ml                                               0.5M Tris, pH 6.8     2.5    ml                                               10% APS               200    μl                                            10% SDS               100    μl                                            TEMED                 12     μl                                            ______________________________________                                    

The gel was poured according to known standard methods and, afteradequate polymerization, fixed in a vertical electrophoresis chamber.For a Coomassie/silver-stained gel, 40 μl each of the A20 sampledescribed under b)=80 μg/4 μg of protein per sample pocket were applied.

The molecular weight standard used was the "Combithek" marker fromBoehringer Mannheim, whose molecular weight range in the reducing samplebuffer extended from 170 to 14 kD.

Composition of the electrophoresis running buffer: ready-to-use dilutionwith Milli Q H₂ O

    ______________________________________                                               SDS           0.1%                                                            Tris          50 mM                                                           Glycine       200 mM                                                   ______________________________________                                    

Flow conditions: about 5 hours at 35 mA/gel (voltage 400 V) when using agel having the measurements 17×18×0.1 cm.

    ______________________________________                                        Stains:                                                                       Sequence                                                                              Time      Composition of the solution                                 ______________________________________                                        1. Coomassie stain                                                            Fixing/ 20-30 min 0.2% Coomassie Brilliant Blue R 250 in                      staining          50% methanol/10% acetic acid/40%                                              H.sub.2 O                                                   Destaining                                                                            as desired,                                                                             20% i-propanol, 7% acetic acid,                                     repeatedly                                                                              3% glycerol, 70% H.sub.2 O                                          change soln.                                                          2. Silver stain (modified Heukeskoven stain)                                  Fixing  30 min    40% ethanol, 10% acetic acid, 50% H.sub.2 O                                   0.40 g of sodium thiosulfate · 5 H.sub.2 O +                         5.00 g of sodium acetate                                    Incubation                                                                            2-24 h    +60 ml of ethanol                                                             shortly before use: + 1.0 ml of glutaralde-                                   hyde (25% strength)                                                           make up to 200 ml with H.sub.2 O                            Washing 3 × 5-10                                                                          H.sub.2 O                                                           min       200 mg of silver nitrate                                    Staining                                                                              45 min    shortly before use: + 40 μl of formaldehyde                                soln., 35% strength                                                           make up to 200 ml with H.sub.2 O                            Washing 10 sec    H.sub.2 O                                                                     5 g of sodium carbonate                                     Developing                                                                            2-10 min  shortly before use: + 20 μl of formaldehyde,                               about 35% strength                                                            make up to 200 ml with H.sub.2 O                            Stopping                                                                              10 min    1.5% strength Na.sub.2 EDTA · 2H.sub.2             ______________________________________                                                          O                                                       

All the abovementioned steps were carried out while gently agitating(shaking table) in 200 ml/gel in each case.

Before photographing/scanning/drying or heat-sealing into plastic bags,the gel was incubated for several hours to overnight in double-distilledH₂ O.

Storage: the heat-sealed gels were stored at room temperature or stackedone on the other in a refrigerator (T:>0° C.), if possible protectedfrom light.

Evaluation/assessment of the gels

In the high-molecular weight range (between marker bands 170 and 116kD), a protein band was detected which is expressed much more stronglyin resistant A20 cells than in A20J.2 cells. This was observed both onCoomassie and on silver staining (see FIG. 1).

Molecular weight

Of the eight calibration standards, the running distance of theindividual proteins in the 4 to 10% gel was plotted in relation to thelogarithm of the molecular weight. It was thus possible to calculate themolecular weight of the abovementioned protein bands having a knownrunning distance. The total running distance was 11.2 cm.

    ______________________________________                                        Protein name of the                                                                           M.sub.r (D)/log M.sub.r                                                                  Running                                            Combithek marker           distance (cm)                                                                            R.sub.f                                 ______________________________________                                        α.sub.2 -macroglobulin                                                                  170000/5.230                                                                             4.37       0.39                                    (equine plasma)                                                               β-galactosidase (E. coli)                                                                116353/5.066                                                                             5.78       0.516                                   Fructose-6-phosphate                                                                          85204/4.930                                                                              7.20       0.643                                   kinase (rabbit muscle)                                                        Glutamate dehydrogenase                                                                       55562/4.745                                                                              8.35       0.746                                   (bovine liver)                                                                Aldolase (rabbit muscle)                                                                      39212/4.593                                                                              9.17       0.819                                   Triose phosphate                                                                              26626/4.425                                                                              10.00      0.893                                   isomerase (rabbit muscle)                                                     Trypsin inhibitor (soybeans)                                                                  20100/4.303                                                                              10.33      0.922                                   Lysozyme (egg white)                                                                          14307/4.156                                                                              10.63      0.949                                   Unknown protein,                                                                              ?          5.58-5.65  0.500                                   5 applications             (5.60)                                             ______________________________________                                    

The mean value of the running distances of the 5 applications of theunknown protein is given in brackets. The correlation coefficientbetween the values was 0.977. The calculated molecular weight is M_(r)135 kDa.

Densitometric evaluation of the quantitative data

On a Bio Image(R) system (Millipore, Eschborn), a quantification of thebands of a Coomassie-stained PAA gel (4 to 10%) with resistant A20 cells(A20R) was performed in using the "whole band menu," as described in themanual for the Bio Image^(R) -System. Result in 5 evaluated trackshaving different protein contents:

    ______________________________________                                        Total amount of protein                                                                      IOD = integrated optical density, (%)                          (μg)        of the 135 kD protein band                                     ______________________________________                                        80             1.07                                                           80             1.03                                                           60             1.05                                                           60             1.04                                                           40             1.32                                                           ______________________________________                                    

Accordingly, the proportion of the 135 kD protein in resistant A20 cellsis about 1% of total cell protein. In normal A20 cells (A20.2 J), it wasnot possible to quantitatively determine this band by applying 80 μg ofprotein, as it was expressed at nearly undetectable levels when comparedto the resistant cells.

Other information was obtained using SDS-PAGE. In one sample work-up,the sample buffer described under a) was modified in that nomercaptoethanol was added. With this condition, the proteins which areformed by S--S bridges do not split up into subunits. Thus, there was nochange in the molecular weight of the 135 kD protein and the proteindoes not likely contain S--S bonds.

d.) Micropreparative concentration of the 135 kDa protein

The amount of protein needed for sequencing is in general given as 100pmol, which corresponds to about 14 μg of protein. On careful estimation(in comparison to the concentration of the marker), the concentration ofthe 135 kDa protein was estimated at 0.3 μg on an 80 μg totalapplication. 16 gels were run (PAA 4 to 10%), which yielded 104 samplesof the 135 kDa bands. These samples were forced through a 32μ sieve. Thetotal amount of protein applied in a sequencing experiment was always 80μg.

e.) Protein digestion in the polyacrylamide gel

After SDS-PAGE and Coomassie staining, the gel bands were excised andwashed until neutral within one day by changing the H₂ O several times.The pieces of gel were then forced through a 32 μm sieve (in a syringewithout a needle). The fine gel paste was then evaporated almost todryness in a vacuum centrifuge.

The addition of enzyme/buffer was then carried out--endoproteinase LYS-C(Boehringer Mannheim) in a 10-fold excess was added. The mixture wasincubated for 6 to 7 hours at 37° C., then eluted at 37° C. for severalhours using 1 ml of 60% acetonitrile/0.1% TFA. The supernatant waspipetted off and the elution was repeated overnight at room temperature.The supernatant was then pipetted off, combined with the firstsupernatant, again filtered through a 0.02 μm filter (Anatop^(R) fromMerck) and evaporated in a vacuum centrifuge.

Before injection into the HPLC, the residue is diluted with 10-20%formic acid.

f.) Peptlide separation the HPLC

    ______________________________________                                        Measuring conditions:                                                         ______________________________________                                        Column:          Superspher ® 60 RP Select B                              Eluent A:        0.1% TFA (trifluoroacetic acid) in                           H.sub.2 O:                                                                    Eluent B:        0.1% TFA in acetonitrile                                     Gradient:        t  min!    % B                                                                0          0                                                                  60         60                                                                 65         70                                                Flow rate:       0.3 ml/min                                                   Measurement wavelength:                                                                        206 nm                                                       ______________________________________                                    

The result is shown in FIG. 2.

g.) Automatic N-terminal protein sequence analysis according to Edmann(Beckmann analyzer; Beckman, Dreieich/Buchschlag, Germany)

    ______________________________________                                        Peptide 1   KLG DI MGVK KE (SEQ ID NO: 1)                                     Peptide 2   KLG DI MGVK KETEPDK (SEQ ID NO: 2)                                Peptide 3   KLIVTSATMDA E K (SEQ ID NO: 3)                                    Peptide 4   DATSDLAIIARK (SEQ ID NO: 4)                                       Peptide 5   KIFQ K (SEQ ID NO: 5)                                             Peptide 6   TP Q EDYV E AAV (SEQ ID NO: 6)                                    ______________________________________                                    

The peaks coresponding to peptides 1 bis 6 are marked in FIG. 2.

h.) Databank comparison with known protein sequences

The peptide sequences obtained in some cases showed a high homology to aprotein derived from the gene sequence of Caenorhabtitis elegans, whosefunction is unknown. The amino acids not corresponding to this C.elegans sequence are underlined (see section g.). The extremely stronghomology of peptide 3 with the C. elegans sequence and the missing orpoor correspondence with peptide 4 or peptide 2 are a clear confirmationof the fact that the protein according to the invention is a novelrepresentative of the DEAD box protein class. It is known from theliterature that the SAT box (corresponding to peptide 3) is highlyconserved in DEAD box proteins from bacteria to mammals.

The following examples describe molecular-biological experiments thatwere carried out. Fundamental molecular-biological standard methods,which are described, for example, in "Molecular Cloning--A LaboratoryManual", 2nd Edition by Sambrook et al., appearing in Cold Spring HarborLaboratory Press, are well known to the skilled artisan. Such techniquesinclude, for example, preparation of plasmid DNA, plasmidminipreparation, plasmid maxipreparation, elution of DNA fragments fromagarose gels, elution by filtration, elution by adsorption, enzymaticmodification of DNA, digestion of the DNA by restriction endonucleases,transformation of E. coli, preparation of RNA, RNA preparation using thesingle-step method (according to Chomzynski), mRNA preparation usingDynabeads^(R), RNA gel electrophoresis, Northern blots, radiolabeling ofDNA, "Random primed" DNA labeling using α-³² P!dATP, sequencing of DNAby the dideoxymethod, cDNA preparation from total RNA, nonradioactivelabeling of nucleic acid, "Random primed" DNA labeling using digoxigenin(DIG), detection of the DIG-labeled nucleic acids.

EXAMPLE 5 PCR Amplification of a cDNA Fragment Corresponding the AminoAcid Sequences from the Murine 135 kD Protein.

The reactions were carried out in a Perkin Elmer cycler. For a 50 μl PCRstandard batch, the following components were pipetted together onto iceand coated with 50 μl of mineral oil:

1 μl of template DNA (0.5-2.5 ng) from A20R cells

1 μl of forward primer (30 pmol/μl)

1 μl of reverse primer (30 pmol/μl)

5 μl of dNTP mixture (2 mM per nucleotide)

5 μl of 10×PCR buffer

36.5 μl of H₂ O

0.5 μl of Taq polymerase (2.5 units)

Amplification was carried out in 40 cycles under the followingconditions:

1st step: Denaturation of the DNA double strand at 94° C., 30 s.

2nd step: Addition of the primer to the DNA single strand at 50° C., 2min.

3rd step: DNA synthesis at 72° C., 3 min.

In the last cycle, the DNA synthesis was carried out for 5 min and thebatch was then cooled down to 4° C. For analysis, 10 μl of the batchwere analyzed on a 1 to 2% strength agarose gel. the following primerswere used:

Forward primer: A20-2, A20-3, A20-4, A20-5 (see FIG. 7)

Reserve primer: A20-6a, A20-6b (see FIG. 7)

Matrices: A20R-total RNA

Forward and reverse primers were in each case combined in pairs in PCRreactions. The batch A20-3/A20-6b led to the amplification of a cDNAabout 630 bp in size, which was reamplified with the primer A20-6b tocheck its specificity with combinations of the primers A20-3, A20-4 andA20-5. To increase the stringency, a temperature of 55° C. was selectedfor the reamplification and only 35 PCR cycles were carried out. Aftercloning and sequencing the fragment obtained (name: A20-5/-6b) usingstandard methods, the sequence data shown in FIG. 8 were obtained. TheA20-5/-6b sequence was obtained by using the primers A20-5 and A20-6b,using A20R DNA as a template. This protein is classified as a DEAH-boxprotein based on the DEAH motif found at amino acids 170-173 of thesequence in FIG. 8 (residue 170-173 of SEQ ID NO:16) and based on thepresence of the amino acid sequences GETGSGKT (residue 73-80 of SEQ IDNO:16) and PRRVAA (residue 103-108 of SEQ ID NO:16), which are alsocharacteristic of DEAH-box proteins.

EXAMPLE 6A Isolation and Sequencing of Full-length Sequence for Murine135 kD DEAH-box Protein

Using techniques that are well known to the skilled artisan, a probe isprepared from the nucleotide sequence in FIG. 8 (SEQ ID NO:15). Forexample, the entire sequence shown in FIG. 8 can be used as a probe.This probe is used to screen a genomic, of cDNA library prepared frommurine A20.2J cells. The full-length clone is isolated a sequenced usingtechniques that are well known to the skilled artisan.

EXAMPLE 7 Northern Hybridization

The hybridization solution used was a ready-to-use ExpressHyb^(R)solution from Clontech, which binds the previously labeled gene probe(radioactive or nonradioactive) to the possibly present complementaryDNA sequence on the carrier filter in a hybridization time of one hour.

Reagents additionally needed:

20×SSC: 3 M NaCl; 0.3 M sodium citrate (pH 7.0)

Wash solution 1: 2×SSC; 0.05% SDS

Wash solution 2: 0.1×SSC; 0.1% SDS

Wash solution 3: 2×SSC; 0.1% SDS

1. Hybridization with nonradiolabeled gene probes using the ExpressHybsolution (Clontech)

The ExpressHyb solution was heated to 68° C. and stirred at the sametime, so that no precipitates remained. The membrane (10×10 cm) was thenprehybridized in at least 5 ml of ExpressHyb solution by mixingcontinuously in a hybridization oven at 68° C. for half an hour. Thenon-radiolabeled DNA probe was mixed with 5 ml of fresh ExpressHybsolution. The prehybridization solution was then replaced by thisExpressHyb® solution and the blot was incubated at 68° C. in thehybridization oven for one hour. After incubation, washing at roomtemperature was carried out for 30 min using 20 ml of the wash solution3 (per 100 cm² of membrane), the solution being replaced once. Thesecond washing step was carried out at 50° C. for 30 min using washsolution 2. Here too, the solution was replaced once. The excess washsolution was then allowed to drip off the membrane and it was thenpossible to use the membrane directly for chemiluminescence detection.

2. Hybridization with radiolabeled gene probes using the ExpressHyb®solution (Clontech)

Hybridization was carried out as in the case of the nonradiolabeled DNAprobe. After incubation, however, washing was carried out with washsolution 1 for 30-40 min at room temperature with replacement of thesolution several times. The second washing step was carried out withwash solution 2 for 40 min at 50° C. In this case, the solution wasreplaced once. After this, the excess wash solution was also allowed todrip off here and the blot was heat-sealed in a plastic film. The blotwas exposed at -70° C. in an exposure cassette or analyzed in aphosphoimager (BIORAD).

The RNA and hybridization probe used are each given in the legends tothe figures.

3. Time course of the mRNA level of the 135 kD protein under theinfluence of leflunomide in A20. J and A20R cells

The experiment is shown in FIGS. 3A and 3B and the associated legend tothe figures. In all cells investigated (A20.2J and A20R) is seen a bandof size 4.4 kilobases. The radiolabeled probe A20-5-6b was used. TheA20R cells give a very strong signal, A20.2J cells only a very weaksignal. However, after treatment of A20.2J cells for one or 8 hours withA77 1226 the band becomes somewhat stronger. A77 1226 does notsignificantly induce the formation of the mRNA investigated here overthe 8 hour time period tested.

4. Time course of the mRNA level of the 135 kD protein with varyingconcentrations of A77 1226.

The experiment is shown in FIGS. 4A and 4B and the associated legend.A20R cells were cultured without A77 1226 in the medium for varyingperiods of time, up to five months. With depletion of A77 1226 overtime, the level of the 4.4 kB mRNA gradually fell. The radiolabeledprobe A20-5/-6b was used.

5. mRNA level of the 135 kD protein in eight different human tissues.

This experiment is described in FIG. 5 and the corresponding legend. Itis seen that the mRNA levels in the tissues investigated are different.The levels of mRNA for the 135 kD protein in heart and skeletal musclewere the highest among the tissues tested. As the mRNA expressioncorrelates with the leflunomide resistance (see Example 3), muscularorgans such as heart and skeletal muscle are possibly less sensitive toleflunomide.

EXAMPLE 8 Homologies of the Murine 135 kD DEAH-box Gene to a Human cDNAClone

The amino acid sequence KLGDIMGVKK (SEQ ID NO:34) from a subregion ofthe differentially expressed 135 Kd DEAH-box protein fromleflunomide-resistant A20R cells was found in an entry of the cDNA cloneB 1 85 (Homo sapiens) in the EM NEW (EMBL-new entries) databank. SeeLiew, etal. Proc. Natl. Acad. Sci. USA 91:10645 (1994). By means ofthis, it was possible to prepare suitable primers for the PCR which wereused in a PCR using a human cDNA bank as a matrix to amplify a cDNAcorresponding to the murine 135 kD protein The novel upstream anddownstream primers are presented in FIG. 7 as primer Nos. 7 and 8(7=hs1, 8=hs2).

The conditions of the PCR were kept stringent, as the primers werecomplementary to the target sequence. The hybridization was carried outfor 45 s at 55° C., the denaturation for 30 s at 94° C. and thesynthesis for only 45 s at 72° C. The reason for the short denaturationand synthesis phase was the known length of the insert to be expected(246 bp). The concentration ratios of the PCR were selected according tostandard as in Example 5. The matrices used were three different humancDNA banks (prepared from (1) peripheral T cells, (2) PMA-stimulatedHI-60 myeloid precursor cells and (3) placenta). In each case a 246bp-long PCR fragment was obtained whose sequence corresponds to thenucleotide 1431 to 1672 of FIG. 9 (bases 1451-1672 of SEQ ID NO:17).

EXAMPLE 9 Obtaining the Complete Human cDNA Clone by ColonyHybridization

On the basis of the results of the Northern blot experiments (Example6), a cDNA bank prepared from human skeletal muscle was used forscreening. The probe employed was the sequence hs1/hs2. This sequencewas prepared using the hs1 and hs2 primers and the Liew, et al. sequenceabove as a template. For the synthesis of labeled probe DNA, hs1/hs2 DNAwas amplified by means of PCR using the primers hsl and hs2 and thehs1/hs2 clone (vector: pCR™II, Invitrogen, San Diego, USA) as a templateand then purified by means of agarose gel electrophoresis and phenolicelution. For DIG labeling with the aid of random primers ("random primedlabeling"), 1 ug of hs1/hs2 DNA was employed as a template and, after areaction time of 20 h, about 2 μg of labeled probe DNA were obtained per1 μg of template. In order to check the probe specificity, a dilutionseries of hs1/hs2 DNA from 0.1 pg to 10 ng was immobilized on nylonmembrane and hybridized with the DIG-labeled hs1/hs2 probe. It was seenthat 5-25 ng of probe per ml of hybridization solution were sufficientin order to detect 10 pg of hs1/hs2 DNA poorly and from 100 pg ofhs1/hs2 DNA (Hybond N+) clearly.

For the first screening of the gene bank, about 40,000 colonies wereplated out per 150 mm agar plate. Altogether, 20 master plates wereprepared, so that about 800,000 individual colonies had been plated out.With this colony count, the probability appeared to be adequate that ina number of 1.1×10⁶ independent clones given by the manufacturer theclone sort was among those plated out. 2 each, i.e. a total of 40replica filters were prepared which were subjected to hybridization withDIG probe. For this hybridization, a probe concentration of 25 ng/ml wasemployed. For detection, the membranes were exposed to X-ray films for 2hours. On 5 different plates a total of 19 positive clones weredetected. Of the 19 positive clones from the primary screening, 5 cloneswere confirmed in the secondary screening. These clones were isolatedand characterized. The following estimated insert sizes resulted for theclones:

    ______________________________________                                                Clone 1                                                                             1.6 kb                                                                  Clone 2                                                                             3.5 kb                                                                  Clone 3                                                                             1.6 kb                                                                  Clone 4                                                                             0.9 kb                                                                  Clone 5                                                                             6.5 kb                                                          ______________________________________                                    

For the purpose of further characterization, the clones were initiallysequenced and the subsequences and restriction maps obtained werecompared with one another. The comparison of the sequences with oneanother confirmed that clone 1 and clone 3 were almost identical. Itturned out that the clones 1, 2, 3 and 4 corresponded to a gene sequencewhich comprised the hs1/hs2 cDNA sequence and corresponded to anestimated length of 4.5 kb. The complete 5'- end and the poly-A tail ofthe mRNA additionally appeared to be contained in this sequence. Fromthe total length, it was expected that this was the complete sequencewhich would be necessary for the expression of a 135 kD protein. Aschematic representation shows the orientation of the human cDNAs to oneanother and the position of the sequence hs1/hs2 used for screening(FIG. 6A).

In comparison to the other clones, clone 5 appeared different. Theinitial sequencing of this clone yielded no overlaps at all with theother sequences and also no indication of the position of the hs1/hs2sequence in the clone. Even in the course of restriction analysis,plasmid 5 showed peculiar features which gave rise to the suspicion thatit did not originate from the same gene as the other clones. Also theunusual length of the inserts, estimated at 6.5 kb, suggested that thisis an isolated cDNA. In other words, this sequence has no overlaps withthe other four clones and is therefore unrelated to those clones. Usingprocedures well known to the skilled artisan, a full length clone isisolated using Clone 5 as a probe. This full-length clone is sequenced.

Clone 1 and clone 2 were completely sequenced. The sequencing data areshown in FIG. 9. The two sequences overlapped by 530 base pairs and whenthe two sequences were superimposed, there was on open reading frame of4.3 kB in length. Clone 1 was exactly 1590 base pairs in length andclone 2 was 3210 base pairs in length. The previously known sequencehs1/hs2 was between positions 1430 and 1672.

The position of this sequence was an indication of the fact that thefirst (beginning with the first base) of the six possible reading frameswas the correct one. In this reading frame were two stop codons: one inbase position 58 (TGA) and one in position 3729 (TGA), after which apoly-A tail followed about 300 base pairs downstream. After the firststop, in position 148 followed a methionine codon which appeared to be apossible start codon for the translation, as it was not only the firstATG codon in the sequence, but also had characteristics of a Kozak startsequence, namely a purine residue (G) in position -3 and a G in position+4. Just under 1000 base pairs further appeared the next ATG codon, moreaccurately two methionine codons in sequence. On account of theenvironment--an A on -3 and a G on +4--the second codon could likewisebe a start codon. As in 90-95% of the cases of known mammalian mRNAtranslation initiation the methionine codon appearing first in thereading frame is simultaneously the start codon, this was also assumedfor the present case. Starting from this assumption, the sequence wouldcode for a 1227 amino acid protein. With an average weight of 110daltons per amino acid, such a protein would correspond to just under135 kD. On account of the size of the protein, the uninterrupted readingframe, and the relatively distinct start codon, the sequence was judgedto be the complete cDNA for the human 135 kD protein.

The similarity of the human sequence with the murine sequence A20-5/6bwas also determined. The comparison of the sequence 05/6b from themurine cell line A20R with the human sequence found yielded a differenceof 15 amino acids out of 245, which corresponded to a percentagedifference of about 6%.

EXAMPLE 10 Homology Domains in the Human Sequence Found and Similaritiesto Other Proteins

Sequence comparison with the homology domains of the superfamily II ofputative helicases showed that all conserved domains of the DEAH proteinfamily were present in the human sequence (FIG. 6b). The followingfeatures distinguish the sequence 135 kD human DEAH-box from otherDEAH-box proteins. First, the first homology domain (ATPase A, Domain I)is located more than 650 amino acids from the N-terminus. Second, thereare Serine/Arginine (SR) domains in the N-terminus. Third, domain IV hasthe sequence FMP instead of FXT. Fourth, the distance between domains IVand V is only 74 amino acids, instead of the 75 to 84 amino acids whichare typical of other DEAH-box proteins. Finally, domain VI has thesequence QRSGRXGR (SEQ ID NO:25) instead of the sequence QRIGRXGR(SEQ IDNO:26).

The first domain--the ATPase A motif, begins with the 655th amino acid.Among domains I through VI, only a two amino acids differ from thehomology sequence for other members of the DEAH-box protein family: aproline instead of a threonine in domain IV and a serine instead of anisoleucine in domain VI. Furthermore, the 654 amino acid distance of thefirst homology domain (Domain I) from the N-terminus is 150 residueslarger than in previously known DEAH box proteins. A further differenceis the distance between domains IV and V: instead of 75 to 80 aminoacids, here there were only 74 amino acid in between these domains inthe gene encoding the human 135 kD DEAH-box protein gene of theinvention.

Otherwise, the protein derived from the human cDNA could be clearlyclassified in the DEAH box proteins family on account of the homologiesshown. In addition, at the N-terminus of the sequence was identified anamino acid sequence which has strong homologies with the "nuclearlocalization site" (NLS) of the SV 40 T antigen. This NLS homologybegins with the 69th amino acid of the human 135 kD protein gene and is10 residues long.

For further characterization of the human 135 kD protein sequence, asequence comparison was carried out in the GCG program with "genembl","swissprot" and "pir" on the DNA and on the protein level.

The gene bank analysis yielded homologies to some already-known proteinsof the DEAH protein family (FIG. 10). The protein with the strongesthomologies was identified as K03H1.2 from C. elegans. This protein wasclassified as a possible DEAH box protein on the basis of homologydomains present (Wilson et al., 1994, Nature 368: 32-38). As indicatedin Example 4(g), originally sequenced peptide fragments of the 135 kDprotein from A20R cells likewise had similarities to the sequence fromC. elegans. This data suggests that the overexpressed protein in A20R isa helicase.

In addition, a protein which was homologous at the DNA level to 60% wasidentified, which was cloned in 1994 from HeLa cells and designated asHRH1 (Ono et al., 1994, Molecular and Cellular Biology. 14: 7611-7620) -likewise a possible human RNA helicase. Further homologies of about 50%at the protein level were found to be the splice factors PRP 2, 16 and22 from S. cerevisiae, likewise members of the DEAH family (Chen andLin, Nucl. Acids Res. 18: 6447, 1990; Schwer und Guthrie, Nature 349:494-499, 1991; Company et al., Nature 349: 487-493, 1991). Furthermore,significant homologies to the DEXH proteins MLE from D. melanogaster(Kuroda et al., 1991, Cell 66: 935-947) and the possible nuclear DNAhelicase 11 - NDH II -from cattle (42 and 43% on the protein level) werefound (Zhang et al., 1995, J. Biol. Chem. 270: 16422-16427).

EXAMPLE 11 In Vitro Expression of the Human 135 kD DEAH-box Protein

By means of rabbit reticulocyte lysate, an in vitro translation of thecDNA obtained was carried out. The full-length 135 kD DEAH-box proteinhuman clone was used. To this end, various batches of linearized andcircular DNA between 0.5 and 2.0 μg were employed. The positive controlused was the luciferase DNA additionally supplied by Promega. Thetranslation was carried out using T7 polymerase. The gene product waslabeled by incorporation of ³⁵ S-methionine and could thus be renderedvisible in an autoradiogram after separation on a denaturing SDS-PAAgel.

Independently of the amount of DNA employed, all batches afforded goodresults, the circular DNA being translated somewhat more efficientlythan the linearized DNA. The positive control showed the expectedluciferase band at 61 kD, the zero control without DNA as expectedafforded no signal. In the gene products of the helicase cDNA, the mostprominent band of synthesized protein with the greatest proteinconcentration was between the protein standards for 97.4 and 220 kD.There were also weaker bands of relatively small translation productswhich were probably formed by the premature termination of protein ormRNA synthesis.

A direct comparison between the native protein from A20R cells and thegene product of the in vivo translation was carried out. To this end,parallel cell lysates of A20.2J and A20R cells and also the in vivotranslation product of the cloned cDNA sequence and the zero controlwere applied to an SDS-PAA gel. As in the 50 μl batch of thereticulocyte lysate, amounts of protein of between 150 and 500 ng areproduced (data from Promega with respect to luciferase control) and1/10th of the batch was applied to a gel pocket. Coomassie staining(bands can be stained from a protein content of 100 ng) was notsufficient to detect the gene product produced in a reticulocyte lysatesystem.

Therefore, in addition to Coomassie staining, an autoradiogram with anX-ray film was set up. It was then possible to apply the film to thedried gel, whereby a direct comparison of the protein bands waspossible. 5 μl of reticulocyte lysate with and without the human 135 kDprotein gene product, 20 μl of A20R lysate and 23 μl of A20.2J lysatewere applied (volumes in each case made up to 30 μl with SDS samplebuffer) to a 7.5% SDS gel (separating gel: 5%). The marker used was a"rainbow marker" and a Coomassie marker. It was seen that a band ofapproximately 135 kD in from the A20R cell lysates appeared in theCoomassie-stained gel. The same gel overlaid with the associatedautoradiogram shows that the band of the full-length 135 kD humanprotein gene product (detected by autoradiography) is at the same heightas the 135 kD protein in A20R.

EXAMPLE 12 Assay of Enzymatic Activity of Human 135 kD DEAH box Protein

The helicase activity of the human 135 kD DEAH box protein is assayedusing a helicase assay technique that is well known in the art, asdescribed in the "Detailed Description of the Invention."

The ATPase activity of the human 135 kD DEAH box protein is assayedusing an ATPase assay technique that is well known in the art asdescribed in the "Detailed Description of the Invention."

The RNA splicing activity of the human 135 kD DEAH box protein isassayed using an RNA splicing assay technique that is well known in theart as described in the "Detailed Description of the Invention."

Priority application 19545126.0, Federal Republic of Germany, filed Dec.4, 1 995, including the specification, drawings, claims and abstract, ishereby incorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 38                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Lys Leu Gly Asp Ile Met Gly Val Lys Lys Gl - #u                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Lys Leu Gly Asp Ile Met Gly Val Lys Lys Gl - #u Thr Glu Pro Asp Lys         #                15                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Lys Leu Ile Val Thr Ser Ala Thr Met Asp Al - #a Glu Lys                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Asp Ala Thr Ser Asp Leu Ala Ile Ile Ala Ar - #g Lys                         #                10                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Lys Ile Phe Gln Lys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Thr Pro Gln Glu Asp Tyr Val Glu Ala Ala Va - #l                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #   17             G                                                          - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 # 20               ARAC                                                       - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                # 20               AYAA                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #  18              GT                                                         - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #  18              AT                                                         - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #             27   CTGC ACTGTCC                                               - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #             27   GAAG GATGCCA                                               - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 612 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..612                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 1..612                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - AAG GAG ACG GAG CCG GAC AAA GCT ATG ACA GA - #A GAC GGG AAA GTG GAC           48                                                                          Lys Glu Thr Glu Pro Asp Lys Ala Met Thr Gl - #u Asp Gly Lys Val Asp           #                 15                                                          - TAC AGG ACG GAG CAG AAG TTT GCA GAT CAC AT - #G AAG GAG AAA AGC GAG           96                                                                          Tyr Arg Thr Glu Gln Lys Phe Ala Asp His Me - #t Lys Glu Lys Ser Glu           #             30                                                              - GCC AGC AGT GAG TTT GCC AAG AAG AAG TCG AT - #C CTG GAG CAG AGG CAG          144                                                                          Ala Ser Ser Glu Phe Ala Lys Lys Lys Ser Il - #e Leu Glu Gln Arg Gln           #         45                                                                  - TAC CTG CCC ATC TTT GCC GTG CAG CAG GAG CT - #C GTC ACC ATC ATC AGA          192                                                                          Tyr Leu Pro Ile Phe Ala Val Gln Gln Glu Le - #u Val Thr Ile Ile Arg           #     60                                                                      - GAC AAC AGC ATT GTG GTC GTG GTC GGG GAG AC - #A GGG AGT GGC AAG ACC          240                                                                          Asp Asn Ser Ile Val Val Val Val Gly Glu Th - #r Gly Ser Gly Lys Thr           # 80                                                                          - ACT CAG CTG ACC CAG TAC TTG CAT GAA GAT GG - #T TAC ACG GAC TAT GGG          288                                                                          Thr Gln Leu Thr Gln Tyr Leu His Glu Asp Gl - #y Tyr Thr Asp Tyr Gly           #                 95                                                          - ATG ATC GGG TGT ACC CAG CCC CGG CGT GTG GC - #T GCC ATG TCA GCG GCC          336                                                                          Met Ile Gly Cys Thr Gln Pro Arg Arg Val Al - #a Ala Met Ser Ala Ala           #           110                                                               - AAG AGA GTC AGT GAA GAG ATG GGG GGC AAC CT - #T GGA GAA GAG GTG GGC          384                                                                          Lys Arg Val Ser Glu Glu Met Gly Gly Asn Le - #u Gly Glu Glu Val Gly           #       125                                                                   - TAT GCC ATC CGC TTT GAG GAC TGC ACT TCG GA - #A AAC AAC TTG ATC AAG          432                                                                          Tyr Ala Ile Arg Phe Glu Asp Cys Thr Ser Gl - #u Asn Asn Leu Ile Lys           #   140                                                                       - TAC ATG ACG GAT GGG ATC CTG CTG CGC GAG TC - #C CTC CGG CAG GCT GAC          480                                                                          Tyr Met Thr Asp Gly Ile Leu Leu Arg Glu Se - #r Leu Arg Gln Ala Asp           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - CTG GAC CAC TAC AGC GCC GTC ATC ATG GAT GA - #G GCC CAC GAG CGC TCC          528                                                                          Leu Asp His Tyr Ser Ala Val Ile Met Asp Gl - #u Ala His Glu Arg Ser           #               175                                                           - CTC AAC ACC GAC GTG CTT TTT GGG CTG CTC CG - #G GAG GTT GTG GCT CGA          576                                                                          Leu Asn Thr Asp Val Leu Phe Gly Leu Leu Ar - #g Glu Val Val Ala Arg           #           190                                                               #      612GAC CTG AAG CTC ATG GTT ACA TCG GC - #T ACT                         Gly Ser Asp Leu Lys Leu Met Val Thr Ser Al - #a Thr                           #       200                                                                   - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 204 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Lys Glu Thr Glu Pro Asp Lys Ala Met Thr Gl - #u Asp Gly Lys Val Asp         #                 15                                                          - Tyr Arg Thr Glu Gln Lys Phe Ala Asp His Me - #t Lys Glu Lys Ser Glu         #             30                                                              - Ala Ser Ser Glu Phe Ala Lys Lys Lys Ser Il - #e Leu Glu Gln Arg Gln         #         45                                                                  - Tyr Leu Pro Ile Phe Ala Val Gln Gln Glu Le - #u Val Thr Ile Ile Arg         #     60                                                                      - Asp Asn Ser Ile Val Val Val Val Gly Glu Th - #r Gly Ser Gly Lys Thr         # 80                                                                          - Thr Gln Leu Thr Gln Tyr Leu His Glu Asp Gl - #y Tyr Thr Asp Tyr Gly         #                 95                                                          - Met Ile Gly Cys Thr Gln Pro Arg Arg Val Al - #a Ala Met Ser Ala Ala         #           110                                                               - Lys Arg Val Ser Glu Glu Met Gly Gly Asn Le - #u Gly Glu Glu Val Gly         #       125                                                                   - Tyr Ala Ile Arg Phe Glu Asp Cys Thr Ser Gl - #u Asn Asn Leu Ile Lys         #   140                                                                       - Tyr Met Thr Asp Gly Ile Leu Leu Arg Glu Se - #r Leu Arg Gln Ala Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Leu Asp His Tyr Ser Ala Val Ile Met Asp Gl - #u Ala His Glu Arg Ser         #               175                                                           - Leu Asn Thr Asp Val Leu Phe Gly Leu Leu Ar - #g Glu Val Val Ala Arg         #           190                                                               - Gly Ser Asp Leu Lys Leu Met Val Thr Ser Al - #a Thr                         #       200                                                                   - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3684 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..3681                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 1..3681                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - ATG GGG GAC ACC AGT GAG GAT GCC TCG ATC CA - #T CGA TTG GAA GGC ACT           48                                                                          Met Gly Asp Thr Ser Glu Asp Ala Ser Ile Hi - #s Arg Leu Glu Gly Thr           #                 15                                                          - GAT CTG GAC TGT CAG GTT GGT GGT CTT ATT TG - #C AAG TCC AAA AGT GCG           96                                                                          Asp Leu Asp Cys Gln Val Gly Gly Leu Ile Cy - #s Lys Ser Lys Ser Ala           #             30                                                              - GCC AGC GAG CAG CAT GTC TTC AAG GCT CCT GC - #T CCC CGC CCT TCA TTA          144                                                                          Ala Ser Glu Gln His Val Phe Lys Ala Pro Al - #a Pro Arg Pro Ser Leu           #         45                                                                  - CTC GGA CTG GAC TTG CTG GCT TCC CTG AAA CG - #G AGA GAG CGA GAG GAG          192                                                                          Leu Gly Leu Asp Leu Leu Ala Ser Leu Lys Ar - #g Arg Glu Arg Glu Glu           #     60                                                                      - AAG GAC GAT GGG GAG GAC AAG AAG AAG TCC AA - #A GTC TCC TCC TAC AAG          240                                                                          Lys Asp Asp Gly Glu Asp Lys Lys Lys Ser Ly - #s Val Ser Ser Tyr Lys           # 80                                                                          - GAC TGG GAA GAG AGC AAG GAT GAC CAG AAG GA - #T GCT GAG GAA GAG GGC          288                                                                          Asp Trp Glu Glu Ser Lys Asp Asp Gln Lys As - #p Ala Glu Glu Glu Gly           #                 95                                                          - GGT GAC CAG GCT GGC CAA AAT ATC CGG AAA GA - #C AGA CAT TAT CGG TCT          336                                                                          Gly Asp Gln Ala Gly Gln Asn Ile Arg Lys As - #p Arg His Tyr Arg Ser           #           110                                                               - GCT CGG GTA GAG ACT CCA TCC CAT CCG GGT GG - #T GTG AGC GAA GAG TTT          384                                                                          Ala Arg Val Glu Thr Pro Ser His Pro Gly Gl - #y Val Ser Glu Glu Phe           #       125                                                                   - TGG GAA CGC AGT CGG CAG AGA GAG CGG GAG CG - #G CGG GAA CAT GGT GTC          432                                                                          Trp Glu Arg Ser Arg Gln Arg Glu Arg Glu Ar - #g Arg Glu His Gly Val           #   140                                                                       - TAT GCC TCG TCC AAA GAA GAA AAG GAT TGG AA - #G AAG GAG AAA TCG CGG          480                                                                          Tyr Ala Ser Ser Lys Glu Glu Lys Asp Trp Ly - #s Lys Glu Lys Ser Arg           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAT CGA GAC TAT GAC CGC AAG AGG GAC AGA GA - #T GAG CGG GAT AGA AGT          528                                                                          Asp Arg Asp Tyr Asp Arg Lys Arg Asp Arg As - #p Glu Arg Asp Arg Ser           #               175                                                           - AGG CAC AGC AGC AGA TCA GAG CGA GAT GGA GG - #G TCA GAG CGT AGC AGC          576                                                                          Arg His Ser Ser Arg Ser Glu Arg Asp Gly Gl - #y Ser Glu Arg Ser Ser           #           190                                                               - AGA AGA AAT GAA CCC GAG AGC CCA CGA CAT CG - #A CCT AAA GAT GCA GCC          624                                                                          Arg Arg Asn Glu Pro Glu Ser Pro Arg His Ar - #g Pro Lys Asp Ala Ala           #       205                                                                   - ACC CCT TCA AGG TCT ACC TGG GAG GAA GAG GA - #C AGT GGC TAT GGC TCC          672                                                                          Thr Pro Ser Arg Ser Thr Trp Glu Glu Glu As - #p Ser Gly Tyr Gly Ser           #   220                                                                       - TCA AGG CGC TCA CAG TGG GAA TCG CCC TCC CC - #G ACG CCT TCC TAT CGG          720                                                                          Ser Arg Arg Ser Gln Trp Glu Ser Pro Ser Pr - #o Thr Pro Ser Tyr Arg           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GAT TCT GAG CGG AGC CAT CGG CTG TCC ACT CG - #A GAT CGA GAC AGG TCT          768                                                                          Asp Ser Glu Arg Ser His Arg Leu Ser Thr Ar - #g Asp Arg Asp Arg Ser           #               255                                                           - GTG AGG GGC AAG TAC TCG GAT GAC ACG CCT CT - #G CCA ACT CCC TCC TAC          816                                                                          Val Arg Gly Lys Tyr Ser Asp Asp Thr Pro Le - #u Pro Thr Pro Ser Tyr           #           270                                                               - AAA TAT AAC GAG TGG GCC GAT GAC AGA AGA CA - #C TTG GGG TCC ACC CCG          864                                                                          Lys Tyr Asn Glu Trp Ala Asp Asp Arg Arg Hi - #s Leu Gly Ser Thr Pro           #       285                                                                   - CGT CTG TCC AGG GGC CGA GGA AGA CGT GAG GA - #G GGC GAA GAA GGA ATT          912                                                                          Arg Leu Ser Arg Gly Arg Gly Arg Arg Glu Gl - #u Gly Glu Glu Gly Ile           #   300                                                                       - TCA TTT GAC ACG GAG GAG GAG CGG CAG CAG TG - #G GAA GAT GAC CAG AGG          960                                                                          Ser Phe Asp Thr Glu Glu Glu Arg Gln Gln Tr - #p Glu Asp Asp Gln Arg           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - CAA GCC GAT CGG GAT TGG TAC ATG ATG GAC GA - #G GGC TAT GAC GAG TTC         1008                                                                          Gln Ala Asp Arg Asp Trp Tyr Met Met Asp Gl - #u Gly Tyr Asp Glu Phe           #               335                                                           - CAC AAC CCG CTG GCC TAC TCC TCC GAG GAC TA - #C GTG AGG AGG CGG GAG         1056                                                                          His Asn Pro Leu Ala Tyr Ser Ser Glu Asp Ty - #r Val Arg Arg Arg Glu           #           350                                                               - CAG CAC CTG CAT AAA CAG AAG CAG AAG CGC AT - #T TCA GCT CAG CGG AGA         1104                                                                          Gln His Leu His Lys Gln Lys Gln Lys Arg Il - #e Ser Ala Gln Arg Arg           #       365                                                                   - CAG ATC AAT GAG GAT AAC GAG CGC TGG GAG AC - #A AAC CGC ATG CTC ACC         1152                                                                          Gln Ile Asn Glu Asp Asn Glu Arg Trp Glu Th - #r Asn Arg Met Leu Thr           #   380                                                                       - AGT GGG GTG GTC CAT CGG CTG GAG GTG GAT GA - #G GAC TTT GAA GAG GAC         1200                                                                          Ser Gly Val Val His Arg Leu Glu Val Asp Gl - #u Asp Phe Glu Glu Asp           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - AAC GCG GCC AAG GTG CAT CTG ATG GTG CAC AA - #T CTG GTG CCT CCC TTT         1248                                                                          Asn Ala Ala Lys Val His Leu Met Val His As - #n Leu Val Pro Pro Phe           #               415                                                           - CTG GAT GGG CGC ATT GTC TTC ACC AAG CAG CC - #G GAG CCG GTG ATT CCA         1296                                                                          Leu Asp Gly Arg Ile Val Phe Thr Lys Gln Pr - #o Glu Pro Val Ile Pro           #           430                                                               - GTG AAG GAT GCT ACT TCT GAC CTG GCC ATC AT - #T GCT CGG AAA GGC AGC         1344                                                                          Val Lys Asp Ala Thr Ser Asp Leu Ala Ile Il - #e Ala Arg Lys Gly Ser           #       445                                                                   - CAG ACA GTG CGG AAG CAC AGG GAG CAG AAG GA - #G CGC AAG AAG GCT CAG         1392                                                                          Gln Thr Val Arg Lys His Arg Glu Gln Lys Gl - #u Arg Lys Lys Ala Gln           #   460                                                                       - CAC AAA CAC TGG GAA CTG GCG GGG ACC AAA CT - #G GGA GAT ATA ATG GGC         1440                                                                          His Lys His Trp Glu Leu Ala Gly Thr Lys Le - #u Gly Asp Ile Met Gly           465                 4 - #70                 4 - #75                 4 -       #80                                                                           - GTC AAG AAG GAG GAA GAG CCA GAT AAA GCT GT - #G ACG GAG GAT GGG AAG         1488                                                                          Val Lys Lys Glu Glu Glu Pro Asp Lys Ala Va - #l Thr Glu Asp Gly Lys           #               495                                                           - GTG GAC TAC AGG ACA GAG CAG AAG TTT GCA GA - #T CAC ATG AAG AGA AAG         1536                                                                          Val Asp Tyr Arg Thr Glu Gln Lys Phe Ala As - #p His Met Lys Arg Lys           #           510                                                               - AGC GAA GCC AGC AGT GAA TTT GCA AAG AAG AA - #G TCC ATC CTG GAG CAG         1584                                                                          Ser Glu Ala Ser Ser Glu Phe Ala Lys Lys Ly - #s Ser Ile Leu Glu Gln           #       525                                                                   - AGG CAG TAC CTG CCC ATC TTT GCA GTG CAG CA - #G GAG CTG CTC ACT ATT         1632                                                                          Arg Gln Tyr Leu Pro Ile Phe Ala Val Gln Gl - #n Glu Leu Leu Thr Ile           #   540                                                                       - ATC AGA GAC AAC AGC ATC GTG ATC GTG GTT GG - #G GAG ACG GGG AGT GGT         1680                                                                          Ile Arg Asp Asn Ser Ile Val Ile Val Val Gl - #y Glu Thr Gly Ser Gly           545                 5 - #50                 5 - #55                 5 -       #60                                                                           - AAG ACC ACT CAG CTG ACG CAG TAC CTG CAT GA - #A GAT GGT TAC ACG GAC         1728                                                                          Lys Thr Thr Gln Leu Thr Gln Tyr Leu His Gl - #u Asp Gly Tyr Thr Asp           #               575                                                           - TAT GGG ATG ATT GGG TGT ACC CAG CCC CGG CG - #T GTA GCT GCC ATG TCA         1776                                                                          Tyr Gly Met Ile Gly Cys Thr Gln Pro Arg Ar - #g Val Ala Ala Met Ser           #           590                                                               - GTG GCC AAG AGA GTC AGT GAA GAG ATG GGG GG - #A AAC CTT GGC GAG GAG         1824                                                                          Val Ala Lys Arg Val Ser Glu Glu Met Gly Gl - #y Asn Leu Gly Glu Glu           #       605                                                                   - GTG GGC TAT GCC ATC CGC TTT GAA GAC TGC AC - #T TCA GAG AAC ACC TTG         1872                                                                          Val Gly Tyr Ala Ile Arg Phe Glu Asp Cys Th - #r Ser Glu Asn Thr Leu           #   620                                                                       - ATC AAA TAC ATG ACT GAC GGG ATC CTG CTC CG - #A GAG TCC CTC CGG GAA         1920                                                                          Ile Lys Tyr Met Thr Asp Gly Ile Leu Leu Ar - #g Glu Ser Leu Arg Glu           625                 6 - #30                 6 - #35                 6 -       #40                                                                           - GCC GAC CTG GAT CAC TAC AGT GCC ATC ATC AT - #G GAC GAG GCC CAC GAG         1968                                                                          Ala Asp Leu Asp His Tyr Ser Ala Ile Ile Me - #t Asp Glu Ala His Glu           #               655                                                           - CGC TCC CTC AAC ACT GAC GTG CTC TTT GGG CT - #G CTC CGG GAG GTA GTG         2016                                                                          Arg Ser Leu Asn Thr Asp Val Leu Phe Gly Le - #u Leu Arg Glu Val Val           #           670                                                               - GCT CGG CGC TCA GAC CTG AAG CTC ATC GTC AC - #A TCA GCC ACG ATG GAT         2064                                                                          Ala Arg Arg Ser Asp Leu Lys Leu Ile Val Th - #r Ser Ala Thr Met Asp           #       685                                                                   - GCG GAG AAG TTT GCT GCC TTT TTT GGG AAT GT - #C CCC ATC TTC CAC ATC         2112                                                                          Ala Glu Lys Phe Ala Ala Phe Phe Gly Asn Va - #l Pro Ile Phe His Ile           #   700                                                                       - CCT GGC CGT ACC TTC CCT GTT GAC ATC CTC TT - #C AGC AAG ACC CCA CAG         2160                                                                          Pro Gly Arg Thr Phe Pro Val Asp Ile Leu Ph - #e Ser Lys Thr Pro Gln           705                 7 - #10                 7 - #15                 7 -       #20                                                                           - GAG GAT TAC GTG GAG GCT GCA GTG AAG CAG TC - #C TTG CAG GTG CAC CTG         2208                                                                          Glu Asp Tyr Val Glu Ala Ala Val Lys Gln Se - #r Leu Gln Val His Leu           #               735                                                           - TCG GGG GCC CCT GGA GAC ATC CTT ATC TTC AT - #G CCT GGC CAA GAG GAC         2256                                                                          Ser Gly Ala Pro Gly Asp Ile Leu Ile Phe Me - #t Pro Gly Gln Glu Asp           #           750                                                               - ATT GAG GTG ACC TCA GAC CAG ATT GTG GAG CA - #T CTG GAG GAA CTG GAG         2304                                                                          Ile Glu Val Thr Ser Asp Gln Ile Val Glu Hi - #s Leu Glu Glu Leu Glu           #       765                                                                   - AAC GCG CCT GCC CTG GCT GTG CTG CCC ATC TA - #C TCT CAG CTG CCT TCT         2352                                                                          Asn Ala Pro Ala Leu Ala Val Leu Pro Ile Ty - #r Ser Gln Leu Pro Ser           #   780                                                                       - GAC CTC CAG GCC AAA ATC TTC CAG AAG GCT CC - #A GAT GGC GTT CGG AAG         2400                                                                          Asp Leu Gln Ala Lys Ile Phe Gln Lys Ala Pr - #o Asp Gly Val Arg Lys           785                 7 - #90                 7 - #95                 8 -       #00                                                                           - TGC ATC GTT GCC ACC AAT ATT GCC GAG ACG TC - #T CTC ACT GTT GAC GGC         2448                                                                          Cys Ile Val Ala Thr Asn Ile Ala Glu Thr Se - #r Leu Thr Val Asp Gly           #               815                                                           - ATC ATG TTT GTT ATC GAT TCT GGT TAT TGC AA - #A TTA AAG GTC TTC AAC         2496                                                                          Ile Met Phe Val Ile Asp Ser Gly Tyr Cys Ly - #s Leu Lys Val Phe Asn           #           830                                                               - CCC AGG ATT GGC ATG GAT GCT CTG CAG ATC TA - #T CCC ATT AGC CAG GCC         2544                                                                          Pro Arg Ile Gly Met Asp Ala Leu Gln Ile Ty - #r Pro Ile Ser Gln Ala           #       845                                                                   - AAT GCC AAC CAG CGG TCA GGG CGA GCC GGC AG - #G ACG GGC CCA GGT CAG         2592                                                                          Asn Ala Asn Gln Arg Ser Gly Arg Ala Gly Ar - #g Thr Gly Pro Gly Gln           #   860                                                                       - TGT TTC AGG CTC TAC ACC CAG AGC GCC TAC AA - #G AAT GAG CTC CTG ACC         2640                                                                          Cys Phe Arg Leu Tyr Thr Gln Ser Ala Tyr Ly - #s Asn Glu Leu Leu Thr           865                 8 - #70                 8 - #75                 8 -       #80                                                                           - ACC ACA GTG CCC GAG ATC CAG AGG ACT AAC CT - #G GCC AAC GTG GTG CTG         2688                                                                          Thr Thr Val Pro Glu Ile Gln Arg Thr Asn Le - #u Ala Asn Val Val Leu           #               895                                                           - CTG CTC AAG TCC CTC GGG GTG CAG GAC CTG CT - #G CAG TTC CAC TTC ATG         2736                                                                          Leu Leu Lys Ser Leu Gly Val Gln Asp Leu Le - #u Gln Phe His Phe Met           #           910                                                               - GAC CCG CCC CCG GAG GAC AAC ATG CTC AAC TC - #T ATG TAT CAG CTC TGG         2784                                                                          Asp Pro Pro Pro Glu Asp Asn Met Leu Asn Se - #r Met Tyr Gln Leu Trp           #       925                                                                   - ATC CTC GGG GCC CTG GAC AAC ACA GGT GGT CT - #G ACC TCT ACC GGG CGG         2832                                                                          Ile Leu Gly Ala Leu Asp Asn Thr Gly Gly Le - #u Thr Ser Thr Gly Arg           #   940                                                                       - CTG ATG GTG GAG TTC CCG CTG GAC CCT GCC CT - #G TCC AAG ATG CTC ATC         2880                                                                          Leu Met Val Glu Phe Pro Leu Asp Pro Ala Le - #u Ser Lys Met Leu Ile           945                 9 - #50                 9 - #55                 9 -       #60                                                                           - GTG TCC TGT GAC ATG GGC TGC AGC TCC GAG AT - #C CTG CTC ATC GTT TCC         2928                                                                          Val Ser Cys Asp Met Gly Cys Ser Ser Glu Il - #e Leu Leu Ile Val Ser           #               975                                                           - ATG CTC TCG GTC CCA GCC ATC TTC TAC AGG CC - #C AAG GGT CGA GAG GAG         2976                                                                          Met Leu Ser Val Pro Ala Ile Phe Tyr Arg Pr - #o Lys Gly Arg Glu Glu           #           990                                                               - GAG AGT GAT CAA ATC CGG GAG AAG TTC GCT GT - #T CCT GAG AGC GAT CAT         3024                                                                          Glu Ser Asp Gln Ile Arg Glu Lys Phe Ala Va - #l Pro Glu Ser Asp His           #      10050                                                                  - TTG ACC TAC CTG AAT GTT TAC CTG CAG TGG AA - #G AAC AAT AAT TAC TCC         3072                                                                          Leu Thr Tyr Leu Asn Val Tyr Leu Gln Trp Ly - #s Asn Asn Asn Tyr Ser           #  10205                                                                      - ACC ATC TGG TGT AAC GAT CAT TTC ATC CAT GC - #T AAG GCC ATG CGG AAG         3120                                                                          Thr Ile Trp Cys Asn Asp His Phe Ile His Al - #a Lys Ala Met Arg Lys           #               10401030 - #                1035                              - GTC CGG GAG GTG CGA GCT CAA CTC AAG GAC AT - #C ATG GTG CAG CAG CGG         3168                                                                          Val Arg Glu Val Arg Ala Gln Leu Lys Asp Il - #e Met Val Gln Gln Arg           #              10550                                                          - ATG AGC CTG GCC TCG TGT GGC ACT GAC TGG GA - #C ATC GTC AGG AAG TGC         3216                                                                          Met Ser Leu Ala Ser Cys Gly Thr Asp Trp As - #p Ile Val Arg Lys Cys           #          10705                                                              - ATC TGT GCT GCC TAT TTC CAC CAA GCA GCC AA - #G CTC AAG GGA ATC GGG         3264                                                                          Ile Cys Ala Ala Tyr Phe His Gln Ala Ala Ly - #s Leu Lys Gly Ile Gly           #      10850                                                                  - GAG TAC GTG AAC ATC CGC ACA GGG ATG CCC TG - #C CAC TTG CAC CCC ACC         3312                                                                          Glu Tyr Val Asn Ile Arg Thr Gly Met Pro Cy - #s His Leu His Pro Thr           #  11005                                                                      - AGC TCC CTT TTT GGA ATG GGC TAC ACC CCA GA - #T TAC ATA GTG TAT CAC         3360                                                                          Ser Ser Leu Phe Gly Met Gly Tyr Thr Pro As - #p Tyr Ile Val Tyr His           #               11201110 - #                1115                              - GAG TTG GTC ATG ACC ACC AAG GAG TAT ATG CA - #G TGT GTG ACC GCT GTG         3408                                                                          Glu Leu Val Met Thr Thr Lys Glu Tyr Met Gl - #n Cys Val Thr Ala Val           #              11350                                                          - GAC GGG GAG TGG CTG GCG GAG CTG GGC CCC AT - #G TTC TAT AGC GTG AAA         3456                                                                          Asp Gly Glu Trp Leu Ala Glu Leu Gly Pro Me - #t Phe Tyr Ser Val Lys           #          11505                                                              - CAG GCG GGC AAG TCA CGG CAG GAG AAC CGT CG - #T CGG GCC AAA GAG GAA         3504                                                                          Gln Ala Gly Lys Ser Arg Gln Glu Asn Arg Ar - #g Arg Ala Lys Glu Glu           #      11650                                                                  - GCC TCT GCC ATG GAG GAG GAG ATG GCG CTG GC - #C GAG GAG CAG CTG CGA         3552                                                                          Ala Ser Ala Met Glu Glu Glu Met Ala Leu Al - #a Glu Glu Gln Leu Arg           #  11805                                                                      - GCC CGG CGG CAG GAG CAG GAG AAG CGC AGC CC - #C CTG GGC AGT GTC AGG         3600                                                                          Ala Arg Arg Gln Glu Gln Glu Lys Arg Ser Pr - #o Leu Gly Ser Val Arg           #               12001190 - #                1195                              - TCT ACG AAG ATC TAC ACT CCA GGC CGG AAA GA - #G CAA GGG GAG CCC ATG         3648                                                                          Ser Thr Lys Ile Tyr Thr Pro Gly Arg Lys Gl - #u Gln Gly Glu Pro Met           #              12150                                                          #     3684CGC CGC ACG CCA GCC CGC TTT GGT CT - #G TGA                         Thr Pro Arg Arg Thr Pro Ala Arg Phe Gly Le - #u                               #           1225                                                              - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1227 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Met Gly Asp Thr Ser Glu Asp Ala Ser Ile Hi - #s Arg Leu Glu Gly Thr         #                 15                                                          - Asp Leu Asp Cys Gln Val Gly Gly Leu Ile Cy - #s Lys Ser Lys Ser Ala         #             30                                                              - Ala Ser Glu Gln His Val Phe Lys Ala Pro Al - #a Pro Arg Pro Ser Leu         #         45                                                                  - Leu Gly Leu Asp Leu Leu Ala Ser Leu Lys Ar - #g Arg Glu Arg Glu Glu         #     60                                                                      - Lys Asp Asp Gly Glu Asp Lys Lys Lys Ser Ly - #s Val Ser Ser Tyr Lys         # 80                                                                          - Asp Trp Glu Glu Ser Lys Asp Asp Gln Lys As - #p Ala Glu Glu Glu Gly         #                 95                                                          - Gly Asp Gln Ala Gly Gln Asn Ile Arg Lys As - #p Arg His Tyr Arg Ser         #           110                                                               - Ala Arg Val Glu Thr Pro Ser His Pro Gly Gl - #y Val Ser Glu Glu Phe         #       125                                                                   - Trp Glu Arg Ser Arg Gln Arg Glu Arg Glu Ar - #g Arg Glu His Gly Val         #   140                                                                       - Tyr Ala Ser Ser Lys Glu Glu Lys Asp Trp Ly - #s Lys Glu Lys Ser Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asp Arg Asp Tyr Asp Arg Lys Arg Asp Arg As - #p Glu Arg Asp Arg Ser         #               175                                                           - Arg His Ser Ser Arg Ser Glu Arg Asp Gly Gl - #y Ser Glu Arg Ser Ser         #           190                                                               - Arg Arg Asn Glu Pro Glu Ser Pro Arg His Ar - #g Pro Lys Asp Ala Ala         #       205                                                                   - Thr Pro Ser Arg Ser Thr Trp Glu Glu Glu As - #p Ser Gly Tyr Gly Ser         #   220                                                                       - Ser Arg Arg Ser Gln Trp Glu Ser Pro Ser Pr - #o Thr Pro Ser Tyr Arg         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Asp Ser Glu Arg Ser His Arg Leu Ser Thr Ar - #g Asp Arg Asp Arg Ser         #               255                                                           - Val Arg Gly Lys Tyr Ser Asp Asp Thr Pro Le - #u Pro Thr Pro Ser Tyr         #           270                                                               - Lys Tyr Asn Glu Trp Ala Asp Asp Arg Arg Hi - #s Leu Gly Ser Thr Pro         #       285                                                                   - Arg Leu Ser Arg Gly Arg Gly Arg Arg Glu Gl - #u Gly Glu Glu Gly Ile         #   300                                                                       - Ser Phe Asp Thr Glu Glu Glu Arg Gln Gln Tr - #p Glu Asp Asp Gln Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gln Ala Asp Arg Asp Trp Tyr Met Met Asp Gl - #u Gly Tyr Asp Glu Phe         #               335                                                           - His Asn Pro Leu Ala Tyr Ser Ser Glu Asp Ty - #r Val Arg Arg Arg Glu         #           350                                                               - Gln His Leu His Lys Gln Lys Gln Lys Arg Il - #e Ser Ala Gln Arg Arg         #       365                                                                   - Gln Ile Asn Glu Asp Asn Glu Arg Trp Glu Th - #r Asn Arg Met Leu Thr         #   380                                                                       - Ser Gly Val Val His Arg Leu Glu Val Asp Gl - #u Asp Phe Glu Glu Asp         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asn Ala Ala Lys Val His Leu Met Val His As - #n Leu Val Pro Pro Phe         #               415                                                           - Leu Asp Gly Arg Ile Val Phe Thr Lys Gln Pr - #o Glu Pro Val Ile Pro         #           430                                                               - Val Lys Asp Ala Thr Ser Asp Leu Ala Ile Il - #e Ala Arg Lys Gly Ser         #       445                                                                   - Gln Thr Val Arg Lys His Arg Glu Gln Lys Gl - #u Arg Lys Lys Ala Gln         #   460                                                                       - His Lys His Trp Glu Leu Ala Gly Thr Lys Le - #u Gly Asp Ile Met Gly         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Lys Lys Glu Glu Glu Pro Asp Lys Ala Va - #l Thr Glu Asp Gly Lys         #               495                                                           - Val Asp Tyr Arg Thr Glu Gln Lys Phe Ala As - #p His Met Lys Arg Lys         #           510                                                               - Ser Glu Ala Ser Ser Glu Phe Ala Lys Lys Ly - #s Ser Ile Leu Glu Gln         #       525                                                                   - Arg Gln Tyr Leu Pro Ile Phe Ala Val Gln Gl - #n Glu Leu Leu Thr Ile         #   540                                                                       - Ile Arg Asp Asn Ser Ile Val Ile Val Val Gl - #y Glu Thr Gly Ser Gly         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Lys Thr Thr Gln Leu Thr Gln Tyr Leu His Gl - #u Asp Gly Tyr Thr Asp         #               575                                                           - Tyr Gly Met Ile Gly Cys Thr Gln Pro Arg Ar - #g Val Ala Ala Met Ser         #           590                                                               - Val Ala Lys Arg Val Ser Glu Glu Met Gly Gl - #y Asn Leu Gly Glu Glu         #       605                                                                   - Val Gly Tyr Ala Ile Arg Phe Glu Asp Cys Th - #r Ser Glu Asn Thr Leu         #   620                                                                       - Ile Lys Tyr Met Thr Asp Gly Ile Leu Leu Ar - #g Glu Ser Leu Arg Glu         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ala Asp Leu Asp His Tyr Ser Ala Ile Ile Me - #t Asp Glu Ala His Glu         #               655                                                           - Arg Ser Leu Asn Thr Asp Val Leu Phe Gly Le - #u Leu Arg Glu Val Val         #           670                                                               - Ala Arg Arg Ser Asp Leu Lys Leu Ile Val Th - #r Ser Ala Thr Met Asp         #       685                                                                   - Ala Glu Lys Phe Ala Ala Phe Phe Gly Asn Va - #l Pro Ile Phe His Ile         #   700                                                                       - Pro Gly Arg Thr Phe Pro Val Asp Ile Leu Ph - #e Ser Lys Thr Pro Gln         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Glu Asp Tyr Val Glu Ala Ala Val Lys Gln Se - #r Leu Gln Val His Leu         #               735                                                           - Ser Gly Ala Pro Gly Asp Ile Leu Ile Phe Me - #t Pro Gly Gln Glu Asp         #           750                                                               - Ile Glu Val Thr Ser Asp Gln Ile Val Glu Hi - #s Leu Glu Glu Leu Glu         #       765                                                                   - Asn Ala Pro Ala Leu Ala Val Leu Pro Ile Ty - #r Ser Gln Leu Pro Ser         #   780                                                                       - Asp Leu Gln Ala Lys Ile Phe Gln Lys Ala Pr - #o Asp Gly Val Arg Lys         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Cys Ile Val Ala Thr Asn Ile Ala Glu Thr Se - #r Leu Thr Val Asp Gly         #               815                                                           - Ile Met Phe Val Ile Asp Ser Gly Tyr Cys Ly - #s Leu Lys Val Phe Asn         #           830                                                               - Pro Arg Ile Gly Met Asp Ala Leu Gln Ile Ty - #r Pro Ile Ser Gln Ala         #       845                                                                   - Asn Ala Asn Gln Arg Ser Gly Arg Ala Gly Ar - #g Thr Gly Pro Gly Gln         #   860                                                                       - Cys Phe Arg Leu Tyr Thr Gln Ser Ala Tyr Ly - #s Asn Glu Leu Leu Thr         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Thr Thr Val Pro Glu Ile Gln Arg Thr Asn Le - #u Ala Asn Val Val Leu         #               895                                                           - Leu Leu Lys Ser Leu Gly Val Gln Asp Leu Le - #u Gln Phe His Phe Met         #           910                                                               - Asp Pro Pro Pro Glu Asp Asn Met Leu Asn Se - #r Met Tyr Gln Leu Trp         #       925                                                                   - Ile Leu Gly Ala Leu Asp Asn Thr Gly Gly Le - #u Thr Ser Thr Gly Arg         #   940                                                                       - Leu Met Val Glu Phe Pro Leu Asp Pro Ala Le - #u Ser Lys Met Leu Ile         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Val Ser Cys Asp Met Gly Cys Ser Ser Glu Il - #e Leu Leu Ile Val Ser         #               975                                                           - Met Leu Ser Val Pro Ala Ile Phe Tyr Arg Pr - #o Lys Gly Arg Glu Glu         #           990                                                               - Glu Ser Asp Gln Ile Arg Glu Lys Phe Ala Va - #l Pro Glu Ser Asp His         #      10050                                                                  - Leu Thr Tyr Leu Asn Val Tyr Leu Gln Trp Ly - #s Asn Asn Asn Tyr Ser         #  10205                                                                      - Thr Ile Trp Cys Asn Asp His Phe Ile His Al - #a Lys Ala Met Arg Lys         #               10401030 - #                1035                              - Val Arg Glu Val Arg Ala Gln Leu Lys Asp Il - #e Met Val Gln Gln Arg         #              10550                                                          - Met Ser Leu Ala Ser Cys Gly Thr Asp Trp As - #p Ile Val Arg Lys Cys         #          10705                                                              - Ile Cys Ala Ala Tyr Phe His Gln Ala Ala Ly - #s Leu Lys Gly Ile Gly         #      10850                                                                  - Glu Tyr Val Asn Ile Arg Thr Gly Met Pro Cy - #s His Leu His Pro Thr         #  11005                                                                      - Ser Ser Leu Phe Gly Met Gly Tyr Thr Pro As - #p Tyr Ile Val Tyr His         #               11201110 - #                1115                              - Glu Leu Val Met Thr Thr Lys Glu Tyr Met Gl - #n Cys Val Thr Ala Val         #              11350                                                          - Asp Gly Glu Trp Leu Ala Glu Leu Gly Pro Me - #t Phe Tyr Ser Val Lys         #          11505                                                              - Gln Ala Gly Lys Ser Arg Gln Glu Asn Arg Ar - #g Arg Ala Lys Glu Glu         #      11650                                                                  - Ala Ser Ala Met Glu Glu Glu Met Ala Leu Al - #a Glu Glu Gln Leu Arg         #  11805                                                                      - Ala Arg Arg Gln Glu Gln Glu Lys Arg Ser Pr - #o Leu Gly Ser Val Arg         #               12001190 - #                1195                              - Ser Thr Lys Ile Tyr Thr Pro Gly Arg Lys Gl - #u Gln Gly Glu Pro Met         #              12150                                                          - Thr Pro Arg Arg Thr Pro Ala Arg Phe Gly Le - #u                             #           1225                                                              - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 43 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Ala Xaa Xaa Xaa Gly Lys Thr Pro Thr Arg Gl - #u Leu Ala Gly Gly Thr         #                15                                                           - Pro Gly Arg Asp Glu Ala Asp Ser Ala Thr Ph - #e Xaa Xaa Xaa Thr Arg         #            30                                                               - Gly Xaa Asp His Arg Ile Gly Arg Xaa Xaa Ar - #g                             #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 43 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Ala Xaa Xaa Xaa Xaa Gly Lys Thr Pro Thr Ar - #g Glu Leu Ala Gly Gly         #                15                                                           - Thr Pro Gly Arg Asp Glu Ala Asp Ser Ala Th - #r Phe Ile Asn Thr Arg         #            30                                                               - Gly Ile Asp His Arg Ile Gly Arg Xaa Xaa Ar - #g                             #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 40 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Gly Xaa Xaa Xaa Xaa Gly Lys Thr Arg Val Al - #a Ala Xaa Xaa Thr Asp         #                15                                                           - Gly Xaa Asp Glu Ala His Ser Ala Thr Phe Xa - #a Thr Xaa Gly Xaa Xaa         #            30                                                               - Gln Arg Ile Gly Arg Xaa Gly Arg                                             #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 37 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Xaa Xaa Xaa Xaa Xaa Gly Lys Thr Pro Thr Ar - #g Xaa Xaa Xaa Asp Glu         #                15                                                           - Xaa His Thr Ala Thr Phe Xaa Xaa Ser Xaa Gl - #y Xaa Xaa Gln Arg Xaa         #            30                                                               - Gly Arg Xaa Gly Arg                                                                 35                                                                    - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Ala Xaa Xaa Xaa Xaa Gly Lys Thr                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - His Arg Ile Gly Arg Xaa Xaa Arg                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Gln Arg Ser Gly Arg Xaa Gly Arg                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Gln Arg Ile Gly Arg Xaa Gly Arg                                             1               5                                                             - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 51 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Glu Asp Lys Lys Lys Ser Lys Val Ser Gly Gl - #u Thr Gly Ser Gly Lys         #                15                                                           - Thr Pro Arg Arg Val Ala Ala Ser Glu Thr As - #p Gly Ile Asp Glu Ala         #            30                                                               - His Ser Ala Thr Phe Met Pro Ser Gly Tyr Cy - #s Gln Arg Ser Gly Arg         #        45                                                                   - Ala Gly Arg                                                                     50                                                                        - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Met Gly Val Lys Lys                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Asp Ile Met Gly Val                                                         1               5                                                             - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Met Gly Val Lys Lys Glu                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Lys Glu Thr Glu Pro Asp                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Thr Ser Ala Thr Met Asp                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Ile Val Thr Ser Ala Thr                                                     1               5                                                             - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Lys Leu Gly Asp Ile Met Gly Val Lys Lys                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Asp Glu Ala Asp                                                             - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Asp Glu Ala His                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Asp Glu Xaa His                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - Asp Glu Xaa Xaa                                                             __________________________________________________________________________

We claim:
 1. An isolated DNA sequence encoding the amino acid sequenceof SEQUENCE ID NO.
 16. 2. An isolated DNA sequence encoding the aminoacid sequence of SEQUENCE ID NO.
 18. 3. An isolated DNA sequence as setforth in SEQUENCE ID NO.
 15. 4. An isolated DNA sequence as set forth inSEQUENCE ID NO.
 17. 5. A polynucleotide that encodes a DEAH-boxleflunomide-resistant protein, and which hybridizes, under stringentconditions, to the polynucleotide of SEQ. ID NO15;wherein said stringentconditions are 60° C. in 0.1×SSC and 0.1% SDS.
 6. A polynucleotide thatencodes a DEAH-box leflunomide-resistant protein, and which hybridizes,under stringent conditions, to the polynucleotide of SEQ. IDNO:17;wherein said stringent conditions are 60° C. in 0.1×SSC and 0.1%SDS.
 7. An expression vector comprising a DNA sequence as claimed inclaim
 5. 8. An expression vector comprising a DNA sequence as claimed inclaim
 6. 9. A process for the preparation of a DEAH-boxleflunomide-resistant protein, wherein said process comprises:(a)culturing a host cell comprising an expression vector according to claim7; and (b) isolating said protein from the cell of step (a).
 10. Aprocess for the preparation of a DEAH-box leflunomide-resistant protein,wherein said process comprises:(a) culturing a host cell comprising anexpression vector according to claim 8; and (b) isolating said proteinfrom the cell of step (a).
 11. An antisense expression vector comprisinga DNA polynucleotide as claimed in claim 5, wherein said polynucleotideis inserted in reverse orientation into said vector.
 12. An antisenseexpression vector comprising a DNA polynucleotide as claimed in claim 6,wherein said polymucleotide is insercted in reverse orientation intosaid vector.